Organic compounds

ABSTRACT

The invention relates to novel inhibitors of phosphodiesterase 1 (PDE1), useful for the treatment of diseases or disorders characterized by disruption of or damage to certain cGMP/PKG mediated pathways (e.g., in cardiac tissue). The invention further relates to pharmaceutical composition comprising the same and methods of treatment of cardiovascular disease and related disorders, e.g., congestive heart disease, atherosclerosis, myocardial infarction, and stroke.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalApplication No. 62/034,567, filed on Aug. 7, 2014, the contents of whichare hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to novel inhibitors of phosphodiesterase 1 (PDE1)useful for the treatment of diseases or disorders characterized bydisruption of or damage to certain cGMP/PKG mediated pathways (e.g., incardiac tissue or in vascular smooth muscle).

The invention further relates to pharmaceutical composition comprisingthe same and methods of treatment of cardiovascular disease and relateddisorders, e.g., congestive heart disease, atherosclerosis, myocardialinfarction, and stroke.

BACKGROUND OF THE INVENTION

Heart disease is a chronic and progressive illness that kills more than2.4 million Americans each year. There are approximately 500,000 newcases of heart failure per year, with an estimated 5 million patients inthe United States alone having this disease. Early intervention islikely to be most effective in preserving cardiac function. It would bemost desirable to prevent as well to reverse the morphological,cellular, and molecular remodeling that is associated with heartdisease. Some of the most important indicators of cardiac risk are age,hereditary factors, weight, smoking, blood pressure, exercise history,and diabetes. Other indicators of cardiac risk include the subject'slipid profile, which is typically assayed using a blood test, or anyother biomarker associated with heart disease or hypertension. Othermethods for assaying cardiac risk include, but are not limited to, anEKG stress test, thallium stress test, EKG, computed tomography scan,echocardiogram, magnetic resonance imaging study, non-invasive andinvasive arteriogram, and cardiac catheterization.

Pulmonary hypertension (PH or PHT) is an increase in blood pressure inthe pulmonary artery, pulmonary vein, and/or pulmonary capillaries. Itis a very serious condition, potentially leading to shortness of breath,dizziness, fainting, decreased exercise tolerance, heart failure,pulmonary edema, and death. It can be one of five different groups,classified by the World Health Organization in categories describedbelow.

WHO Group I—Pulmonary Arterial Hypertension (PAH):

-   -   a. Idiopathic (IPAH)    -   b. Familial (FPAH)    -   c. Associated with other diseases (APAH): collagen vascular        disease (e.g. scleroderma), congenital shunts between the        systemic and pulmonary circulation, portal hypertension, HIV        infection, drugs, toxins, or other diseases or disorder.    -   d. Associated with venous or capillary disease.

Pulmonary arterial hypertension involves the vasoconstriction ortightening of blood vessels connected to and within the lungs. Thismakes it harder for the heart to pump blood through the lungs, much asit is harder to make water flow through a narrow pipe as opposed to awide one. Over time, the affected blood vessels become both stiffer andthicker, in a process known as fibrosis. This further increases theblood pressure within the lungs and impairs their blood flow. Inaddition, the increased workload of the heart causes thickening andenlargement of the right ventricle, making the heart less able to pumpblood through the lungs, causing right heart failure. As the bloodflowing through the lungs decreases, the left side of the heart receivesless blood. This blood may also carry less oxygen than normal.Therefore, it becomes more and more difficult for the left side of theheart to pump to supply sufficient oxygen to the rest of the body,especially during physical activity.

WHO Group II—Pulmonary Hypertension Associated with Left Heart Disease:

-   -   a. Atrial or ventricular disease    -   b. Valvular disease (e.g. mitral stenosis)

In pulmonary hypertension WHO Group II, there may not be any obstructionto blood flow in the lungs. Instead, the left heart fails to pump bloodefficiently out of the heart into the body, leading to pooling of bloodin veins leading from the lungs to the left heart (congestive heartfailure or CHF). This causes pulmonary edema and pleural effusions. Thefluid build-up and damage to the lungs may also lead to hypoxia andconsequent vasoconstriction of the pulmonary arteries, so that thepathology may come to resemble that of Group I or III.

WHO Group III—Pulmonary Hypertension Associated with Lung Diseasesand/or Hypoxemia:

-   -   a. Chronic obstructive pulmonary disease (COPD), interstitial        lung disease (ILD)    -   b. Sleep-disordered breathing, alveolar hypoventilation    -   c. Chronic exposure to high altitude    -   d. Developmental lung abnormalities

In hypoxic pulmonary hypertension (WHO Group III), the low levels ofoxygen may cause vasoconstriction or tightening of pulmonary arteries.This leads to a similar pathophysiology as pulmonary arterialhypertension.

WHO Group IV—Pulmonary Hypertension Due to Chronic Thrombotic and/orEmbolic Disease:

-   -   a. Pulmonary embolism in the proximal or distal pulmonary        arteries    -   b. Embolization of other matter, such as tumor cells or        parasites

In chronic thromboembolic pulmonary hypertension (WHO Group IV), theblood vessels are blocked or narrowed with blood clots. Again, thisleads to a similar pathophysiology as pulmonary arterial hypertension.

WHO Group V—Miscellaneous

Treatment of pulmonary hypertension has proven very difficult.Antihypertensive drugs that work by dilating the peripheral arteries arefrequently ineffective on the pulmonary vasculature. For example,calcium channel blockers are effective in only about 5% of patients withIPAH. Left ventricular function can often be improved by the use ofdiuretics, beta blockers, ACE inhibitors, etc., or by repair/replacementof the mitral valve or aortic valve. Where there is pulmonary arterialhypertension, treatment is more challenging, and may include lifestylechanges, digoxin, diuretics, oral anticoagulants, and oxygen therapy areconventional, but not highly effective. Newer drugs targeting thepulmonary arteries, include endothelin receptor antagonists (e.g.,bosentan, sitaxentan, ambrisentan), phosphodiesterase type 5 inhibitors(e.g., sildenafil, tadalafil), prostacyclin derivatives (e.g.,epoprostenol, treprostenil, iloprost, beroprost), and soluble guanylatecyclase (sGC) activators (e.g., cinaciguat and riociguat). Surgicalapproaches to PAH include atrial septostomy to create a communicationbetween the right and left atria, thereby relieving pressure on theright side of the heart, but at the cost of lower oxygen levels in blood(hypoxia); lung transplantation; and pulmonary thromboendarterectomy(PTE) to remove large clots along with the lining of the pulmonaryartery. Heart failure and acute myocardial infarction are common andserious conditions frequently associated with thrombosis and/or plaquebuild-up in the coronary arteries.

Cardiovascular disease or dysfunction may also be associated withdiseases or disorders typically thought of as affecting skeletal muscle.One such disease is Duchenne muscular dystrophy (DMD), which is adisorder that primarily affects skeletal muscle development but can alsoresult in cardiac dysfunction and cardiomyopathy. DMD is a recessiveX-linked form of muscular dystrophy, affecting around 1 in 3,600 boys,which results in muscle degeneration and eventual death. The disorder iscaused by a mutation in the dystrophin gene, located on the human Xchromosome, which codes for the protein dystrophin, an importantstructural component within muscle tissue that provides structuralstability to the dystroglycan complex (DGC) of the cell membrane. Whileboth sexes can carry the mutation, females rarely exhibit signs of thedisease.

Patients with DMD either lack expression of the protein dystrophin orexpress inappropriately spliced dystrophin, as a result of mutations inthe X-linked dystrophin gene. Additionally, the loss of dystrophin leadsto severe skeletal muscle pathologies as well as cardiomyopathy, whichmanifests as congestive heart failure and arrhythmias. The absence of afunctional dystrophin protein is believed to lead to reduced expressionand mis-localization of dystrophin-associated proteins includingNeuronal Nitric Oxide (NO) Synthase (nNOS). Disruption of nNOS signalingmay result in muscle fatigue and unopposed sympathetic vasoconstrictionduring exercise, thereby increasing contraction-induced damage indystrophin-deficient muscles. The loss of normal nNOS signaling duringexercise is central to the vascular dysfunction proposed to be animportant pathogenic mechanism in DMD. Eventual loss of cardiac functionoften leads to heart failure in DMD patients.

Currently, there is a largely unmet need for an effective way oftreating cardiovascular disease and disorders (e.g. congestive heartdisease), and diseases and disorders which may result in cardiacdysfunction or cardiomyopathy (e.g., Duchenne Muscular Dystrophy).Improved therapeutic compounds, compositions and methods for thetreatment of cardiac conditions and dysfunction are urgently required.

Eleven families of phosphodiesterases (PDEs) have been identified butonly PDEs in Family I, the Ca²⁺-calmodulin-dependent phosphodiesterases(CaM-PDEs), which are activated by the Ca²⁺-calmodulin and have beenshown to mediate the calcium and cyclic nucleotide (e.g. cAMP and cGMP)signaling pathways. The three known CaM-PDE genes, PDE1A, PDE1B, andPDE1C, are all expressed in central nervous system tissue. PDE1A isexpressed throughout the brain with higher levels of expression in theCA1 to CA3 layers of the hippocampus and cerebellum and at a low levelin the striatum. PDE1A is also expressed in the lung and heart. PDE1B ispredominately expressed in the striatum, dentate gyrus, olfactory tractand cerebellum, and its expression correlates with brain regions havinghigh levels of dopaminergic innervation. Although PDE1B is primarilyexpressed in the central nervous system, it is also detected in theheart, is present in neutrophils and has been shown to be involved ininflammatory responses of this cell. PDE1C is expressed in olfactoryepithelium, cerebellar granule cells, striatum, heart, and vascularsmooth muscle. PDE1C is a major phosphodiesterase in the human cardiacmyocyte.

Of all of the PDE families, the major PDE activity in the human cardiacventricle is PDE1. Generally, there is a high abundance of PDE1 isoformsin: cardiac myocytes, vascular endothelial cells, vascular smooth musclecells, fibroblasts and motor neurons. Up-regulation of phosphodiesterase1A expression is associated with the development of nitrate tolerance.Kim et al., Circulation 104(19:2338-2343 (2001). Cyclic nucleotidephosphodiesterase IC promotes human arterial smooth muscle cellproliferation. Rybalkin et al., Circ. Res. 90(2):151-157 (2002). Thecardiac ischemia-reperfusion rat model also shows an increase in PDE1activity. Kakkar et al., can. J. Physiol. Pharmacol. 80(1):59-66 (2002).Ca²⁺/CaM-stimulated PDE1, particularly PDE1A has been shown to beinvolved in regulating pathological cardiomyocyte hypertrophy. Millet etal., Circ. Res. 105(10):956-964 (2009). Early cardiac hypertrophyinduced by angiotensin II is accompanied by 140% increases in PDE1A in arat model of heart failure. Mokni et al., Plos. One. 5(12):e14227(2010). Inhibition of phosphodiesterase 1 augments the pulmonaryvasodilator response to inhaled nitric oxide in awake lambs with acutepulmonary hypertension. Evgenov et al., Am. J. Physiol. Lung Cell. Mol.Physiol. 290(4):L723-L729 (2006). Strong upregulation of the PDE1 familyin pulmonary artery smooth muscle cells is also noted in humanidiopathic PAH lungs and lungs from animal models of PAH. Schermuly etal., Circulation 115(17)2331-2339 (2007). PDE1A and IC, found infibroblasts, are known to be up-regulated in the transition to the“synthetic phenotype”, which is connected to the invasion of diseasedheart tissue by pro-inflammatory cells that will deposit extracellularmatrix. PDE1B2, which is present in neutrophils, is up-regulated duringthe process of differentiation of macrophages. Bender et al., PNAS102(2):497-502 (2005). The differentiation of monocytes to macrophage,in turn, is involved in the inflammatory component of heart disease,particularly atherothrombosis, the underlying cause of approximately 80%of all sudden cardiac death. Willerson et al., Circulation109:II-2-II-10 (2004).

Cyclic nucleotide phosphodiesterases downregulate intracellular cAMP andcGMP signaling by hydrolyzing these cyclic nucleotides to theirrespective 5′-monophosphates (5′AMP and 5′GMP). cGMP is a centralintracellular second-messenger regulating numerous cellular functions.In the cardiac myocyte, cGMP mediates the effects of nitric oxide andatrial natriuretic peptide, whereas its counterpart, cAMP, mediatescatecholamine signaling. Each cyclic nucleotide has a correspondingprimary targeted protein kinase, PKA for cAMP, and PKG for cGMP. PKAstimulation is associated with enhanced contractility and can stimulategrowth, whereas PKG acts as a brake in the heart, capable of counteringcAMP-PKA-contractile stimulation and inhibiting hypertrophy.Importantly, the duration and magnitude of these signaling cascades aredetermined not only by generation of cyclic nucleotides, but also bytheir hydrolysis catalyzed by phosphodiesterases (PDEs). PDE regulationis quite potent—often suppressing an acute rise in a given cyclicnucleotide back to baseline within seconds. It is also compartmentalizedwithin the cell, so that specific targeted proteins can be regulated bythe same “generic” cyclic nucleotide. By virtue of its modulation ofcGMP in the myocyte, PDE1 participates in hypertrophy regulation. (CircRes. 2009, Nov. 6; 105(10):931).

One of the challenges currently faced in the field is the lack of PDE1specific inhibitors. The current invention seeks to overcome this aswell as other challenges in the art by providing PDE1 specificinhibitors. Although WO 2006/133261 and WO 2009/075784 provide PDE1specific inhibitors, these do not disclose the compounds of the currentinvention.

SUMMARY OF THE INVENTION

PDE1 is up-regulated in chronic disease conditions such asatherosclerosis, cardiac pressure-load stress and heart failure, as wellas in response to long-term exposure to nitrates. PDE1 inhibitors haverelatively little impact on resting function, but rather maintain theability to potently modulate acute contractile tone in cells stimulatedby vasoactive agonists. Such up-regulation contributes to vascular andcardiac pathophysiology and to drug tolerance to nitrate therapies.Therefore, without being bound by theory, it is believed that compoundsthat modulate cGMP/PKG mediated pathways, such as PDE1 inhibitors, areparticularly useful for reversing cardiac hypertrophy. The PDE1inhibitors disclosed herein are selective PDE1 inhibitors having alimited ability to penetrate the blood brain barrier and therefore, arebelieved to have significant modulatory activity (e.g., enhancement ofcGMP) in those areas of the body outside of the central nervous systemwhere PDE1 isoforms are predominately located: e.g., cardiac, vascular,and lung tissue.

Therefore, in the first aspect, the invention provides a compound ofFormula I:

in free or salt form, wherein

-   (i) R₁ is C₁₋₄alkyl (e.g., methyl or ethyl), or —NH(R₂), wherein R₂    is phenyl optionally substituted with halo (e.g., fluoro), for    example, 4-fluorophenyl;-   (ii) X, Y and Z are, independently, N or C;-   (iii) R₃, R₄ and R₅ are independently H or C₁₋₄alkyl (e.g., methyl);    or R₃ is H and R₄ and R₅ together form a tri-methylene bridge (pref.    wherein the R₄ and R₅ together have the cis configuration, e.g.,    where the carbons carrying R₄ and R₅ have the R and S    configurations, respectively),-   (iv) R₆, R₇ and R₈ are independently:    -   H,    -   C₁₋₄alkyl (e.g., methyl),    -   pyrid-2-yl substituted with hydroxy, or    -   —S(O)₂—NH₂;-   (v) Provided that when X, Y and/or Z are N, then R₆, R₇ and/or R₈,    respectively, are not present; and when X, Y and Z are all C, then    at least one of R₆, R₇ or R₈ is —S(O)₂—NH₂ or pyrid-2-yl substituted    with hydroxy.

In a particular embodiment, the invention provides a compound of FormulaI as follows:

-   -   1.1 the compound of Formula I, wherein R₁ is C₁₋₄alkyl (e.g.,        methyl or ethyl) or —NH(R₂) wherein R₂ is phenyl optionally        substituted with halo (e.g., fluoro), for example,        4-fluorophenyl;    -   1.2 the compound of Formula I, wherein R₁ is C₁₋₄alkyl (e.g.,        methyl or ethyl);    -   1.3 the compound of Formula I, wherein R₁ is —NH(R₂), wherein R₂        is phenyl optionally substituted with halo (e.g., fluoro);    -   1.4 the compound of Formula I, wherein R₁ is —NH(R₂), wherein R₂        is phenyl;    -   1.5 the compound of Formula I, wherein R₁ is —NH(R₂), wherein R₂        is phenyl substituted with halo (e.g., fluoro), for example,        4-fluorophenyl;    -   1.6 the compound of Formula I or any of formulae 1.1-1.5,        wherein X, Y and Z are, independently, N or C;    -   1.7 the compound of Formula I or any of formulae 1.1-1.5,        wherein X, Y and Z are all C;    -   1.8 the compound of Formula I or any of formulae 1.1-1.5,        wherein at least one of X, Y and/or Z is N;    -   1.9 the compound of Formula I or any of formulae 1.1-1.5,        wherein X is N;    -   1.10 the compound of Formula I or any of formulae 1.1-1.5,        wherein Z is N;    -   1.11 the compound of Formula I or any of formulae 1.1-1.5,        wherein Y is N;    -   1.12 the compound of Formula I or any of formulae 1.1-1.5,        wherein X and Z are N and Y is C;    -   1.13 the compound of Formula I or any of formulae 1.1-1.12,        wherein R₆, R₇ and R₈ are independently:        -   H,        -   C₁₋₄alkyl (e.g., methyl),        -   pyrid-2-yl substituted with hydroxy, or        -   —S(O)₂—NH₂;    -   1.14 the compound of Formula I or any of formulae 1.1-1.12,        wherein one or more of R₆, R₇ and R₈ are independently H;    -   1.15 the compound of Formula I or any of formulae 1.1-1.12,        wherein R₆, R₇ and R₈ are independently C₁₋₄alkyl (e.g.,        methyl);    -   1.16 formula 1.15, provided that when R₆, R₇ and/or R₈ are        independently C₁₋₄alkyl (e.g., methyl), then X, Y, and/or Z,        respectively, are C (or not N);    -   1.17 the compound of Formula I or any of formulae 1.1-1.12,        wherein R₆, R₇ and R₈ are independently H or C₁₋₄alkyl (e.g.,        methyl);    -   1.18 the compound of Formula I or any of formulae 1.1-1.12,        wherein R₆, R₇ and R₈ are independently pyrid-2-yl substituted        with hydroxy;    -   1.19 the compound of Formula I or any of formulae 1.1-1.12,        wherein R₇ is pyrid-2-yl substituted with hydroxyl (e.g,        6-hydroxypyrid-2-yl);    -   1.20 formula 1.18 or 1.19, provided that when R₆, R₇ and/or R₈        are independently pyrid-2-yl substituted with hydroxy, then X, Y        and/or Z, respectively are independently C (or not N);    -   1.21 the compound of Formula I or any of formulae 1.1-1.12,        wherein one or more of R₆, R₇ and R₈ are independently        —S(O)₂—NH₂;    -   1.22 the compound of Formula I or 1.7, wherein at least one of        R₆, R₇ and R₈ is —S(O)₂—NH₂ or pyrid-2-yl substituted with        hydroxy;    -   1.23 the compound of Formula I or 1.9, wherein R₆ does not exist        and R₇ and R₈ are H;    -   1.24 the compound of Formula I or 1.10, wherein R₈ does not        exist and R₆ and R₇ are H;    -   1.25 the compound of Formula I or 1.11, wherein R₇ does not        exist and R₆ and R₈ are H;    -   1.26 the compound of Formula I or 1.12, wherein R₆ and R₈ do not        exist and R₇ is C₁₋₄alkyl (e.g., methyl);    -   1.27 the compound of Formula I or 1.12, wherein R₆ and R₈ do not        exist and R₇ is methyl;    -   1.28 the compound of Formula I or any of formula 1.1-1.27,        wherein R₃, R₄ and R₅ are independently H or C₁₋₄alkyl; or R₃ is        H and R₄ and R₅ together form a tri-methylene bridge (pref.        wherein the R₄ and R₅ together have the cis configuration, e.g.,        where the carbons carrying R₄ and R₅ have the R and S        configurations, respectively);    -   1.29 the compound of Formula I or any of formula 1.1-1.27,        wherein R₃, R₄ and R₅ are independently H or C₁₋₄alkyl;    -   1.30 the compound of Formula I or any of formula 1.1-1.27,        wherein R₃ and R₄ are independently C₁₋₄alkyl and R₅ is H;    -   1.31 the compound of Formula I or any of formula 1.1-1.27,        wherein R₃ and R₄ are both methyl and R₅ is H;    -   1.32 the compound of Formula I or any of formula 1.1-1.27,        wherein R₃ is H and R₄ and R₅ together form a tri-methylene        bridge (pref. wherein the R₄ and R₅ together have the cis        configuration, e.g., where the carbons carrying R₄ and R₅ have        the R and S configurations, respectively);    -   1.33 the compound of formula 1.32, wherein the R₄ and R₅        together have the cis configuration, e.g., where the carbons        carrying R₄ and R₅ have the R and S configurations,        respectively);    -   1.34 the compound of Formula I, wherein:        -   (i) R₁ is —N(R₂), wherein R₂ is phenyl optionally            substituted with halo (e.g., fluoro), for example,            4-fluorophenyl;        -   (ii) At least one of X, Y and Z is N;        -   (iii) R₃, R₄ and R₅ are independently H or C₁₋₄alkyl; or R₃            is H and R₄ and R₅ together form a tri-methylene bridge            (pref. wherein the R₄ and R₅ together have the cis            configuration, e.g., where the carbons carrying R₄ and R₅            have the R and S configurations, respectively),        -   (iv) R₆, R₇ and R₈ are independently H or C₁₋₄alkyl (e.g.,            methyl);    -   1.35 the compound of Formula I or 1.34, wherein R₃, R₄ and R₅        are independently H or C₁₋₄alkyl (e.g., methyl);    -   1.36 the compound of Formula I or 1.34 or 1.35, wherein R₃ and        R₄ are independently C₁₋₄alkyl (e.g., R₃ and R₄ are methyl), and        R₅ is H;    -   1.37 the compound of Formula I, wherein:        -   R₁ is —N(R₂), wherein R₂ is phenyl optionally substituted            with halo (e.g., fluoro), for example, 4-fluorophenyl;        -   X and Z are N and Y is C;        -   R₇ is C₁₋₄alkyl (e.g., methyl);        -   R₃ and R₄ are C₁₋₄alkyl (e.g., methyl);        -   R₅ is H;    -   1.38 the compound according to any of the above formulae,        selected from the group consisting of:

-   3-((4-fluorophenyl)amino)-5,7,7-trimethyl-2-((2-methylpyrimidin-5-yl)methyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one;

-   3-((4-fluorophenyl)amino)-5,7,7-trimethyl-2-(pyridin-3-ylmethyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one;

-   3-((4-fluorophenyl)amino)-5,7,7-trimethyl-2-(pyridin-4-ylmethyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one;

-   (6aR,9aS)-2-(4-(6-hydroxypyridin-2-yl)benzyl)-5-methyl-3-(phenylamino)-5,6a,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one;

-   4-(((6aR,9aS)-5-methyl-4-oxo-3-(phenylamino)-4,5,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-2(6aH)-yl)methyl)benzenesulfonamide;

-   3-((4-fluorophenyl)amino)-5,7,7-trimethyl-2-((6-methylpyridin-3-yl)methyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one;

-   4-((3-((4-fluorophenyl)amino)-5,7,7-trimethyl-4-oxo-4,5,7,8-tetrahydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-2-yl)methyl)benzenesulfonamide;

-   3-((4-fluorophenyl)amino)-2-(4-(6-hydroxypyridin-2-yl)benzyl)-5,7,7-trimethyl-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one;

-   2-(4-(6-hydroxypyridin-2-yl)benzyl)-3,5,7,7-tetramethyl-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one;

-   3-ethyl-2-(4-(6-hydroxypyridin-2-yl)benzyl)-5,7,7-trimethyl-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one;    -   1.39 any of the preceding formulae wherein the compounds inhibit        phosphodiesterase-mediated (e.g., PDE1-mediated) hydrolysis of        cGMP, e.g., with an IC₅₀ of less than 1 μM, preferably less than        75 nM, preferably less than 1 nM, in an immobilized-metal        affinity particle reagent PDE assay, for example, as described        in Example 11;        in free or salt form, provided that when X, Y and/or Z are N,        then R₆, R₇ and/or R₈, respectively, are not present; and when        X, Y and Z are all C, then at least one of R₆, R₇ or R₈ is        —S(O)₂—NH₂ or pyrid-2-yl substituted with hydroxy.

In the second aspect, the invention provides a pharmaceuticalcomposition comprising the compound of Formula I or any of 1.1-1.39 asdescribed herein, in free or pharmaceutically acceptable salt form, inadmixture (or in combination or association) with a pharmaceuticallyacceptable diluents or carrier.

The compound of Formula I or any of 1.1-1.39 as described herein areselective PDE1 inhibitors and therefore are useful for regulatingcGMP/PKG in cardiac hypertrophy. Previous studies have demonstrated thatincreases in intracellular Ca²⁺/CaM-dependent signaling promotemaladaptive hypertrophic gene expression in cardiomyocytes throughvarious effectors such as the protein phosphatase calcineurin,Ca²⁺/CaM-dependent kinase II (CaMKII). Without being bound by anytheory, increases in endogenous cGMP/PKG-dependent signaling may be ableto decrease cardiac hypertrophy, by suppressing Gq/11 activation andnormalizing Ca²⁺ signaling. By activating PDE1, Ca²⁺/CaM may decreasecGMP levels and PKG activity. In turn, this process may drive cardiachypertrophy. Additionally, up-regulation of PDE1 expression uponneurohumoral or biomechanical stress during cardiac hypertrophy mayfurther enhance PDE1 activity and attenuate cGMP/PKG signaling.Accordingly, without being bound by any theory, it is believed thatinhibition of PDE1A could, for example, reverse or prevent theattenuation of cGMP/PKG signaling. As discussed previously, PDE1B isalso implicated in the inflammatory component of heart disease (e.g.,PDE1B2 is up-regulated during the process of differentiation ofmacrophages, as occurs during heart disease progression). Similarly,PDE1C is induced in human arterial smooth muscle cells of the synthetic,proliferative phenotype. Therefore, administration of a PDE1 inhibitoras described herein could provide a potential means to regulate cardiachypertrophy, and by extension provide a treatment for variouscardiovascular diseases and disorders.

Therefore, in the third aspect, the invention provides a method for thetreatment or prophylaxis of a disease or disorder which may beameliorated by modulating (e.g., enhancing) cGMP/PKG-dependent signalingpathways (e.g., in cardiac tissue), e.g. a cardiovascular disease ordisorder, comprising administering to a patient in need thereof aneffective amount of the compound of Formula I or any of formulae1.1-1.39 as described herein, in free or pharmaceutically acceptablesalt form.

The cardiovascular disease or disorder may be selected from a groupconsisting of: hypertrophy (e.g., cardiac hypertrophy), atherosclerosis,myocardial infarction, congestive heart failure, angina, stroke,hypertension, essential hypertension, pulmonary hypertension, pulmonaryarterial hypertension, secondary pulmonary hypertension, isolatedsystolic hypertension, hypertension associated with diabetes,hypertension associated with atherosclerosis, renovascular hypertension.In certain embodiments, the cardiovascular disease or disorder to betreated may also relate to impaired cGMP/PKG-dependent signaling. In aparticular embodiment, the invention provides a method for the treatmentor prevention of stroke, wherein the PDE1 inhibitor, through its effecton the endothelial cells of cerebral capillaries, is able to promoteincreased cerebral blood flow.

In a further embodiment of the third aspect, the invention also providesa method for the treatment or prophylaxis of cardiovascular disease ordisorder that is associated with a muscular dystrophy (e.g, Duchenne,Becker, limb-girdle, myotonic, and Emery-Dreifuss Muscular Dystrophy)comprising administering to a patient in need thereof an effectiveamount of the compound of Formula I or any of 1.1-1.39 as describedherein, in free or pharmaceutically acceptable salt form. As notedabove, DMD is caused by the absence of a functional dystrophin protein,which in turn leads to reduced expression and mis-localization ofdystrophin-associated proteins, such as neuronal nitric oxide (NO)synthase. Disruption of nNOS signaling may result in muscle fatigue andunopposed sympathetic vasoconstriction during exercise, therebyincreasing contraction-induced damage in dystrophin-deficient muscles.Without being bound by theory, the loss of normal nNOS signaling duringexercise may be central to the vascular dysfunction proposed to be animportant pathogenic mechanism in DMD. It is contemplated that byinhibiting phosphodiesterases (e.g. PDE1), the compounds describedherein may circumvent defective nNOS signaling in dystrophic skeletaland/or cardiac muscle; thereby potentially improving cardiac outcomes inDMD patients.

In still another embodiment, the invention provides for the treatment ofrenal failure, fibrosis, inflammatory disease or disorders, vascularremodeling and connective tissue diseases or disorders (e.g., MarfanSyndrome), comprising administering to a patient in need thereof aneffective amount of the compound of Formula I or any of formulae1.1-1.39 as described herein, in free or pharmaceutically acceptablesalt form.

The PDE1 compounds of the invention useful for the treatment orprophylaxis of disease according to the foregoing methods may be used asa sole therapeutic agent or may be used in combination with one or moreother therapeutic agents useful for the treatment of cardiovasculardisorders. Such other agents include angiotensin II receptorantagonists, angiotensin-converting-enzyme (ACE) inhibitors, neutralendopeptidase (NEP or Neprilysin) inhibitors and/or phosphodiesterase 5(PDE5) inhibitors.

Therefore, in a particular embodiment, the PDE1 inhibitor of theinvention may be administered in combination with an angiotensin IIreceptor antagonist selected from azilsartan, candesartan, eprosartan,irbesartan, losartan, olmesartan, olmesartan medoxomil, saralasin,telmisartan and valsartan, in free or pharmaceutically acceptable saltform.

In yet another embodiment, the PDE1 inhibitor of the invention may beadministered in combination with an angiotensin-converting-enzyme (ACE)inhibitor selected from the group consisting of: captopril, enalapril,lisinopril, benazapril, ramipril, quinapril, peridopril, imidapril,trandolapril and cilazapril, in free or pharmaceutically acceptable saltform.

In still another particular embodiment, the PDE1 inhibitor of theinvention may be administered in combination with a PDE5 inhibitorselected from avanafil, lodenafil, mirodenafil, tadalafil, vardenafil,udenafil and zaprinast, in free or pharmaceutically acceptable saltform.

In still another particular embodiment, the PDE1 inhibitor of theinvention may be administered in combination with a neutralendopeptidase (NEP or Neprilysin) inhibitor. Neutral endopeptidase, alsoknown as Neprilysin or NEP (EC 3.4.24.11), is a type II integralmembrane zinc-dependent metalloendoprotease that cleaves a variety ofshort peptide substrates. In mammals, NEP is widely expressed, includingin the kidney, lung, endothelial cells, vascular smooth muscle cells,cardiac myocytes, fibroblasts, adipocytes and brain. Among its naturaltargets are cardiac atrial natriuretic peptide (ANP), B-type natriureticpeptide (BNP), C-type natriuretic peptide (CNP), angiotensin I (Ang-I),angiotensin II (Ang-II), bradykinin (BK), and endothelin (ET). Cleavageof these peptides by NEP results in their inactivation, attenuating thepeptides' natural biological effects.

ANP, BNP and CNP are all part of the natriuretic peptide (NP) system,which, along with the renin-angiotensin system, is a major components ofmammalian blood pressure homeostasis. While the renin-angiotensin systemis primarily responsible for increasing blood pressure (e.g., bypromoting vasoconstriction and water retention), the natriuretic peptidesystem is primarily responsible for decreasing blood pressure (e.g., bypromoting vasodilation and natriuresis). ANP and BNP are both powerfulvasodilators and strong promoters of decreased renal reabsorption ofsodium and water in a potassium-sparing manner. These dual effects exerta powerful blood pressure lowering effect. BNP and CNP also exert ananti-fibrotic effect and an anti-hypertrophic effect in the heart. CNPshares the vasodilatory effects of ANP/BNP but without the renaleffects. In addition, both hypertension and obesity have been shown tobe associated with reduced ANP and BNP levels, and a specific geneticvariant of ANP (rs5068), which increases ANP levels, has been shown toprotect against hypertension and metabolic syndrome. Thus, ANP, BNP andCNP play an important role in blood pressure homeostasis andcardiovascular health.

Inhibition of NEP results in an increase in the half-lives ofcirculating ANP, BNP and CNP. This is expected to prolong theirblood-pressure lowering and cardiac health improving effects. Urine cAMPlevels are significantly elevated after systemic administration of NEPinhibitors.

Inhibition of NEP also results in higher levels of bradykinin,angiotensin I, angiotensin II and endothelin. Importantly, endothelinand angiotensin II are strongly pro-hypertensive peptides. Thus, NEPinhibition alone results in both vasodilatory effects (from the NPs) andvasoconstrictive effects (from increased Ang-II and ET). Thesepro-hypertensive peptides all operate via binding to G-protein coupledreceptors (GPCRs). The major contributor to this vasoconstrictive effectis Angiotensin-II, which operates via binding to the G-protein coupledreceptors AT₁ and AT₂. These receptors exert their physiological effectsthrough activation of phospholipase C (PLC) and protein kinase C (PKC)signaling cascades. Bradykinin is inactivated to a large extent by ACE,and ACE inhibitors cause congestion as a major side effect, which is notseen with NEP inhibitors.

ANP, BNP and CNP all function via the second messenger cGMP. ANP and BNPbind to membrane-bound guanylyl cyclase-A, while CNP binds to guanylylcyclase B. Both of these enzymes increase intracellular cGMP in responseto receptor binding. The increased cGMP concentration activates proteinkinase G (PKG) which is responsible for exerting the downstreambiological effects of the natriuretic peptides.

Several NEP inhibitors are known, including candoxatril, candoxatrilat,omepatrilat, gempatrilat, and sampatrilat. Candoxatril had been shown toproduce a dose-dependent increase in both plasma ANP and cGMP levels,and although it is safe, it does not produce a stable blood-pressurelowering effect. This is thought to be due to the effects of NEPinhibition on BK, ET and Ang-II breakdown. Candoxatril treatment inpatients with heart failure has been shown to increase levels ofendothelin significantly, thus cancelling out the blood pressure effectscaused by increased ANP.

In contrast to candoxatril and candoxatrilat, omapatrilat is considereda vasopeptidase inhibitor (VPI), because it functions to an equal extentas both an NEP inhibitor and an ACE (angiotensin converting enzyme)inhibitor. ACE is the enzyme that is responsible for converting Ang-I toAng-II, which is the major pro-hypertensive hormone of therenin-angiotensin system. By inhibiting both NEP and ACE, it was thoughtthat the increase in Ang-II caused by NEP inhibition would be negated,resulting in a highly effective antihypertensive treatment. Clinicalstudies, however, showed that omapatrilat was associated with a severeincidence of angioedema (a known side effect of ACE inhibitors). Laterresearch has indicated that this may be due to concomitant inhibition ofaminopeptidase P (APP). ACE, APP and NEP all contribute to the breakdownof bradykinin, which is another anti-hypertensive peptide, and theover-accumulation of bradykinin resulting from simultaneous inhibitionof three of its degradation pathways may be a strong factor leading toangioedema.

Without intending to be bound by any particular theory, the combinationof a PDE1 inhibitor with a selective NEP inhibitor (not a VPI) shouldrealize the full positive effects of NEP inhibition (increased ANP, BNPand CNP half-life), further enhanced by the potentiation of the NPsignaling cascades (mediated by cGMP) caused by PDE1 inhibition, withoutthe negative effects of NEP inhibition that can lead to decreasedefficacy.

Therefore, in a particular embodiment, the neutral endopeptidase (NEP orNeprilysin) inhibitor useful for the invention is a selective NEPinhibitor, e.g., with at least 300-fold selectivity for NEP inhibitionover ACE inhibition. In a further embodiment, the NEP inhibitors for usein the current invention are inhibitors with at least 100-foldselectivity for NEP inhibition over ECE (Endothelin Converting Enzyme)inhibition. In yet another embodiment, the NEP inhibitors for use in thecurrent invention are inhibitors with at least 300-fold selectivity forNEP inhibition over ACE inhibition and 100-fold selectivity for NEPinhibition over ECE inhibition.

In another embodiment, the NEP inhibitors for use in the currentinvention are the NEP inhibitors disclosed in the followingpublications: EP-1097719 B1, EP-509442A, U.S. Pat. No. 4,929,641,EP-599444B, US-798684, J. Med. Chem. (1993) 3821, EP-136883, U.S. Pat.No. 4,722,810, Curr. Pharm. Design (1996) 443, J. Med. Chem. (1993) 87,EP-830863, EP-733642, WO 9614293, WO 9415908, WO 9309101, WO 9109840,EP-519738, EP-690070, Bioorg. Med. Chem. Lett. (1996) 65, EP-A-0274234,Biochem. Biophys. Res. Comm. (1989) 58, Perspect. Med. Chem. (1993) 45,or EP-358398-B. The contents of these patents and publications arehereby incorporated by reference in their entirety herein.

In still another embodiment, the NEP inhibitors useful in the currentinvention are the NEP inhibitors Phosphoramidon, Thiorphan,Candoxatrilat, Candoxatril, or the compound of the Chemical AbstractService (CAS) Number 115406-23-0.

In yet another embodiment, the NEP inhibitors useful in the currentinvention are the NEP inhibitors disclosed in US 2006/0041014 A1, thecontents of which are hereby incorporated by reference in their entiretyherein.

In another embodiment, the NEP inhibitors useful in the currentinvention are the NEP inhibitors disclosed in U.S. Pat. No. 5,217,996,the contents of which are hereby incorporated by reference in theirentirety herein.

In another embodiment, the NEP inhibitors useful in the currentinvention are the NEP inhibitors disclosed in U.S. Pat. No. 8,513,244,the contents of which are hereby incorporated by reference in theirentirety herein.

In another embodiment, the NEP inhibitors useful in the currentinvention are the NEP inhibitors disclosed in U.S. Pat. No. 5,217,996,the contents of which are hereby incorporated by reference in theirentirety herein.

In another embodiment, the NEP inhibitors useful in the currentinvention are the NEP inhibitors disclosed in US patent applicationpublication 2013/0330365, the contents of which are hereby incorporatedby reference in their entirety herein.

In another embodiment, the NEP inhibitor useful in the current inventionis3-[{1S,5R}-1-biphenyl-4ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl]propionicacid,

also known as AHU-377, in free or a pharmaceutically acceptable salt orprodrug, thereof, and in a preferred embodiment thereof, in sodium saltform.

In another embodiment, the NEP inhibitor useful in the current inventionis [2R, 4R}-1-biphenyl-4ylmethyl-3-carboxy-1-butylcarbamoyl]propionicacid,

also known as LBQ-657, in free or pharmaceutically acceptable ester,salt or prodrug form.

In another embodiment, the NEP inhibitor useful in the current inventionis selected from among the following: sampatrilat, fasidotril, Z13752A,MDL 100240, BMS 189921, LBQ657, AHU-377, or mixanpril, in free orpharmaceutically acceptable salt form or in prodrug form thereof.

In another embodiment, the NEP inhibitor for use in the currentinvention is selected from the following:

-   SQ 28,603;-   N—[N-[1(S)-carboxyl-3-phenylpropyl]-(S)-phenylalanyl]-(S)-isoserine;-   N—[N-[((1S)-carboxy-2-phenyl)ethyl]-(S)-phenylalanyl]-beta-alanine;-   N-[2(S)-mercaptomethyl-3-(2-methylphenyl)-propionyl]methionine;-   (cis-4-[[[1-[2-carboxy-3-(2-methoxy-ethoxy)propyl]-cyclopentyl]carbonyl]amino]-cyclohexanecarboxylic    acid);-   thiorphan; retro-thiorphan; phosphoramidon; SQ 29072;-   N-(3-carboxy-1-oxopropyl)-(4S)-p-phenylphenylmethyl)-4-amino-2R-methylbutanoic    acid ethyl ester;-   (S)-cis-4-[1-[2-(5-indanyloxycarbonyl)-3-(2-methoxyethoxy)propyl]-1-cyclopentanecarboxamido]-1-cyclohexanecarboxylic    acid;-   3-(1-[6-endo-hydroxymethyl-bicyclo[2,2,1]heptane-2-exo-carbamoyl]cyclopentyl)-2-(2-methoxyethyl)propanoic    acid;-   N-(1-(3-(N-t-butoxycarbonyl-(S)-prolylamino)-2(S)-t-butoxy-carbonylpropyl)cyclopentanecarbonyl)-O-benzyl-(S)-serine    methyl ester;-   4-[[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]amino]benzoic acid;-   3-[1-(cis-4-carboxycarbonyl-cis-3-butylcyclohexyl-r-1-carbamoyl)cyclopentyl]-2S-(2-methoxyethoxymethyl)propanoic    acid;-   N-((2S)-2-(4-biphenylmethyl)-4-carboxy-5-phenoxyvaleryl)glycine;-   N-(1-(N-hydroxycarbamoylmethyl)-1-cyclopentanecarbonyl)-L-phenylalanine;-   (S)-(2-biphenyl-4-yl)-1-(1H-tetrazol-5-yl)ethylamino)methylphosphonic    acid;-   (S)-5-(N-(2-(phosphonomethylamino)-3-(4-biphenyl)propionyl)-2-aminoethyl)tetrazole;-   beta-alanine;-   3-[1,1′-biphenyl]-4-yl-N-[diphenoxyphosphinyl)methyl]-L-alanyl;-   N-(2-carboxy-4-thienyl)-3-mercapto-2-benzylpropanamide;-   2-(2-mercaptomethyl-3-phenylpropionamido)thiazol-4-ylcarboxylic    acid;-   (L)-(1-((2,2-dimethyl-1,3-dioxolan-4-yl)-methoxy)carbonyl)-2-phenylethyl)-L-phenylalanyl)-beta-alanine;-   N—[N-[(L)-[1-[(2,2-dimethyl-1,3-dioxolan-4-yl)-methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-(R)-alanine;-   N—[N-[(L)-1-carboxy-2-phenylethyl]-L-phenylalanyl]-(R)-alanine;-   N-[2-acetylthiomethyl-3-(2-methyl-phenyl)propionyl]-methionine ethyl    ester;-   N-[2-mercaptomethyl-3-(2-methylphenyl)propionyl]-methionine;-   N-[2(S)-mercaptomethyl-3-(2-methylphenyl)propanoyl]-(S)-isoserine;-   N—(S)-[3-mercapto-2-(2-methylphenyl)propionyl]-(S)-2-methoxy-(R)-alanine;-   N-[1-[[1(S)-benzyloxy-carbonyl-3-phenylpropyl]amino]cyclopentylcarbony    1]-(S)-isoserine;-   N-[1-[[1(S)-carbonyl-3-phenylpropyl]amino]-cyclopentylcarbonyl]-(S)-isoserine;-   1,1′-[dithiobis-[2(S)-(2-methylbenzyl)-1-oxo-3,1-propanediyl]]-bis-(S)-isoserine;-   1,1′-[dithiobis-[2(S)-(2-methylbenzyl)-1-oxo-3,    1-propanediyl]]-bis-(S)-methionine;-   N-(3-phenyl-2-(mercaptomethyl)-propionyl)-(S)-4-(methylmercapto)methionine;-   N-[2-acetylthiomethyl-3-phenyl-propionyl]-3-aminobenzoic acid;-   N-[2-mercaptomethyl-3-phenyl-propionyl]-3-aminobenzoic acid;-   N-[1-(2-carboxy-4-phenylbutyl)-cyclopentane-carbonyl]-(S)-isoserine;-   N-[1-(acetylthiomethyl)cyclopentane-carbonyl]-(S)-methionine ethyl    ester;-   3(S)-[2-(acetylthiomethyl)-3-phenyl-propionyl]amino-epsilon-caprolactam;-   N-(2-acetylthiomethy 1-3-(2-methylphenyl)propionyl)-methionine ethyl    ester;    in free or pharmaceutically acceptable salt form.

In another aspect, the invention provides the following:

-   -   (i) the compound of Formula I or any of 1.1-1.39 as described        herein, in free or pharmaceutically acceptable salt form, for        use in any of the methods or in the treatment or prophylaxis of        any disease or disorder as set forth herein,    -   (ii) a combination as described hereinbefore, comprising a PDE1        inhibitor of the invention, e.g., the compound of Formula I or        any of 1.1-1.39 as described herein, in free or pharmaceutically        acceptable salt form and a second therapeutic agent useful for        the treatment of cardiovascular disorders, e.g., selected from        angiotensin II receptor antagonist,        angiotensin-converting-enzyme (ACE) inhibitor, neutral        endopeptidase (NEP or Neprilysin) inhibitor and/or        phosphodiesterase 5 (PDE5) inhibitor, in free or        pharmaceutically acceptable salt form;    -   (iii) use of the compound of Formula I or any of 1.1-1.39, in        free or pharmaceutically acceptable salt form, or the        combination described herein, (in the manufacture of a        medicament) for the treatment or prophylaxis of any disease or        condition as set forth herein,    -   (iv) the compound of Formula I or any of 1.1-1.39, in free or        pharmaceutically acceptable salt form, the combination described        herein or the pharmaceutical composition of the invention as        hereinbefore described for use in the treatment or prophylaxis        of any disease or condition as set forth herein.

DETAILED DESCRIPTION OF THE INVENTION

If not otherwise specified or clear from context, the following termsherein have the following meanings:

-   -   (a) “Alkyl” as used herein is a saturated or unsaturated        hydrocarbon moiety, preferably saturated, preferably having one        to six carbon atoms, in some embodiment, one to four carbon        atoms, which may be linear or branched, and may be optionally        mono-, di- or tri-substituted, e.g., with halogen (e.g., chloro        or fluoro) or hydroxy.

The term “optionally substituted with” is intended to be eithersubstituted with a substituent or unsubstituted. In one embodiment, theinvention is substituted. In another embodiment, the invention isunsubstituted.

Compounds of the Invention, e.g., the compound of Formula I or any offormulae 1.1-1.39 as described herein, may exist in free or salt form,e.g., as acid addition salts. In this specification unless otherwiseindicated, language such as “Compounds of the Invention” is to beunderstood as embracing the compounds in any form, for example free oracid addition salt form, or where the compounds contain acidicsubstituents, in base addition salt form. The Compounds of the Inventionare intended for use as pharmaceuticals, therefore pharmaceuticallyacceptable salts are preferred. Salts which are unsuitable forpharmaceutical uses may be useful, for example, for the isolation orpurification of free Compounds of the Invention or theirpharmaceutically acceptable salts, are therefore also included.

Compounds of the Invention may in some cases exist in prodrug form. Aprodrug form is compound which converts in the body to a Compound of theInvention. For example when the Compounds of the Invention containhydroxy or carboxy substituents, these substituents may formphysiologically hydrolysable and acceptable esters. As used herein,“physiologically hydrolysable and acceptable ester” means esters ofCompounds of the Invention which are hydrolysable under physiologicalconditions to yield acids (in the case of Compounds of the Inventionwhich have hydroxy substituents) or alcohols (in the case of Compoundsof the Invention which have carboxy substituents) which are themselvesphysiologically tolerable at doses to be administered. Therefore,wherein the Compound of the Invention contains a hydroxy group, forexample, Compound-OH, the acyl ester prodrug of such compound, i.e.,Compound-O—C(O)—C₁₋₄alkyl, can hydrolyze in the body to formphysiologically hydrolysable alcohol (Compound-OH) on the one hand andacid on the other (e.g., HOC(O)—C₁₋₄alkyl). Alternatively, wherein theCompound of the Invention contains a carboxylic acid, for example,Compound-C(O)OH, the acid ester prodrug of such compound,Compound-C(O)O—C₁₋₄alkyl can hydrolyze to form Compound-C(O)OH andHO—C₁₋₄alkyl. As will be appreciated the term thus embraces conventionalpharmaceutical prodrug forms.

The Compounds of the Invention include their enantiomers, diastereomersand racemates, as well as their polymorphs, hydrates, solvates andcomplexes. Some individual compounds within the scope of this inventionmay contain double bonds. Representations of double bonds in thisinvention are meant to include both the E and the Z isomer of the doublebond. In addition, some compounds within the scope of this invention maycontain one or more asymmetric centers. This invention includes the useof any of the optically pure stereoisomers as well as any combination ofstereoisomers.

It is also intended that the Compounds of the Invention encompass theirstable and unstable isotopes. Stable isotopes are nonradioactiveisotopes which contain one additional neutron compared to the abundantnuclides of the same species (i.e., element). It is expected that theactivity of compounds comprising such isotopes would be retained, andsuch compound would also have utility for measuring pharmacokinetics ofthe non-isotopic analogs. For example, the hydrogen atom at a certainposition on the Compounds of the Invention may be replaced withdeuterium (a stable isotope which is non-radioactive). Examples of knownstable isotopes include, but not limited to, deuterium, ¹³C, ¹⁵N, ¹⁸O.Alternatively, unstable isotopes, which are radioactive isotopes whichcontain additional neutrons compared to the abundant nuclides of thesame species (i.e., element), e.g., ¹²³I, ¹³¹I, ¹²⁵I, ¹¹C, ¹⁸F, mayreplace the corresponding abundant species of I, C and F. Anotherexample of useful isotope of the compound of the invention is the ¹¹Cisotope. These radio isotopes are useful for radio-imaging and/orpharmacokinetic studies of the compounds of the invention.

Melting points are uncorrected and (dec) indicates decomposition.Temperature are given in degrees Celsius (° C.); unless otherwisestated, operations are carried out at room or ambient temperature, thatis, at a temperature in the range of 18-25° C. Chromatography meansflash chromatography on silica gel; thin layer chromatography (TLC) iscarried out on silica gel plates. NMR data is in the delta values ofmajor diagnostic protons, given in parts per million (ppm) relative totetramethylsilane (TMS) as an internal standard. Conventionalabbreviations for signal shape are used. Coupling constants (J) aregiven in Hz. For mass spectra (MS), the lowest mass major ion isreported for molecules where isotope splitting results in multiple massspectral peaks Solvent mixture compositions are given as volumepercentages or volume ratios. In cases where the NMR spectra arecomplex, only diagnostic signals are reported.

Methods of Using Compounds of the Invention

The Compounds of the Invention are useful in the treatment of diseasescharacterized by disruption of or damage to cGMP/PKG mediated pathways,e.g., as a result of increased expression of PDE1 or decreasedexpression of cGMP/PKG activity due to inhibition or reduced levels ofinducers of cyclic nucleotide synthesis, such as dopamine and nitricoxide (NO). It is believed that by inhibiting PDE1A or PDE1C, forexample, that this action could reverse or prevent the attenuation ofcGMP/PKG signaling (e.g., enhance cGMP) and that this action couldmodulate cardiac hypertrophy. Therefore, administration or use of thePDE1 inhibitor as described herein, e.g., a Compound of Formula 1.1-1.39as described herein, in free or pharmaceutically acceptable salt form,could provide a potential means to regulate cardiac hypertrophy (e.g.,prevent and/or reverse cardiac hypertrophy), and in certain embodimentsprovide a treatment for various cardiovascular diseases and disorders.

The PDE1 inhibitors of the present invention generally are selective forPDE1 (generally off-target interactions >10×, more preferably >25×,still more preferably >100× affinity for PDE1), exhibit good oralavailability in plasma with very minimal brain penetration in mice. Theblood/plasma ratio in mice administered the PDE1 inhibitors of thepresent invention is preferably less than 0.4, more preferably less than0.2, more preferably less than or equal to 0.1.

The PDE1 inhibitor of the invention (i.e., Formula I as hereinbeforedescribed) may be used in a combination therapy wherein the PDE1inhibitor may be administered simultaneously, separately or sequentiallywith another active agent. Therefore, the combination can be a freecombination or a fixed combination.

The term “simultaneously” when referring to a therapeutic use meansadministration of two or more active ingredients at or about the sametime by the same route of administration.

The term “separately” when referring to a therapeutic use meansadministration of two or more active ingredients at or about the sametime by different route of administration.

The dosages of a compound of the invention in combination with anotheractive agent can be the same as or lower than the approved dosage forthe drug, the clinical or literature test dosage or the dosage used forthe drug as a monotherapy. The dosage amount will be apparent to oneskilled in the art.

Diseases and disorders that may be prevented or ameliorated by theenhancement of cGMP/PKG signaling (e.g., cardiovascular disease), e.g.,using the Compound of the Invention as described herein, include, butare not limited to: hypertrophy (e.g., cardiac hypertrophy),atherosclerosis, myocardial infarction, congestive heart failure,angina, stroke, hypertension, essential hypertension, pulmonaryhypertension, pulmonary arterial hypertension, secondary pulmonaryhypertension, isolated systolic hypertension, hypertension associatedwith diabetes, hypertension associated with atherosclerosis,renovascular hypertension, renal failure, fibrosis, an inflammatorydisease or disorder, vascular remodeling, and an connective tissuedisease or disorder (e.g., Marfan Syndrome).

In one embodiment, the Compounds of the Invention as described hereinare useful in the treatment or prevention of stroke by treating orpreventing transient ischemic attacks (TIA). Without being bound by anytheory, it is believed that the Compounds of the Invention may preventor treat the risk of transient ischemic attacks by actually increasingthe amount and/or concentration of blood flow to the brain. It iscontemplated that the compounds as described herein could increase theblood flow to the brain without significant passage across the bloodbrain barrier.

In another embodiment, the invention further provides using theCompounds of the Invention for the treatment or prevention of disease ordisorder as follows: Duchenne muscular dystrophy, Becker musculardystrophy, limb-girdle muscular dystrophy, myotonic dystrophy, andEmery-Dreifuss muscular dystrophy. In one embodiment, the Compounds ofthe Invention are useful in treating cardiac dysfunction associated withaforementioned types of muscular dystrophy. In another embodiment, theCompounds of the Invention may potentially reduce or reverse the cardiachypertrophy that may be associated with these aforementioned types ofmuscular dystrophy.

“PDE1 inhibitor” as used herein describes a compound of Formula I or anyof formulae 1.1-1.39 which selectively inhibitphosphodiesterase-mediated (e.g., PDE1-mediated, especiallyPDE1B-mediated) hydrolysis of cGMP, e.g., with an IC₅₀ of less than 1μM, preferably less than 75 nM, preferably less than 1 nM, in animmobilized-metal affinity particle reagent PDE assay as described orsimilarly described in Example 1.

The phrase “Compounds of the Invention” or “PDE 1 inhibitors of theInvention”, or like terms, encompasses any and all of the compoundsdisclosed herewith, e.g., a Compound of Formula I or any of formulae1.1-1.39, in free or salt form.

The words “treatment” and “treating” are to be understood accordingly asembracing treatment or amelioration of symptoms of disease as well astreatment of the cause of the disease.

For methods of treatment, the word “effective amount” is intended toencompass a therapeutically effective amount to treat a specific diseaseor disorder.

The term “patient” include human or non-human (i.e., animal) patient. Inparticular embodiment, the invention encompasses both human andnonhuman. In another embodiment, the invention encompasses nonhuman. Inother embodiment, the term encompasses human.

The term “comprising” as used in this disclosure is intended to beopen-ended and does not exclude additional, unrecited elements or methodsteps.

Compounds of the Invention, e.g., compounds of Formula I or any offormulae 1.1-1.39 as hereinbefore described, in free or pharmaceuticallyacceptable salt form, may be used as a sole therapeutic agent, but mayalso be used in combination or for co-administration with other activeagents.

Dosages employed in practicing the present invention will of course varydepending, e.g. on the particular disease or condition to be treated,the particular compound used, the mode of administration, and thetherapy desired. The compound may be administered by any suitable route,including orally, parenterally, transdermally, or by inhalation, but arepreferably administered orally. In general, satisfactory results, e.g.for the treatment of diseases as hereinbefore set forth are indicated tobe obtained on oral administration at dosages of the order from about0.01 to 2.0 mg/kg. In larger mammals, for example humans, an indicateddaily dosage for oral administration will accordingly be in the range offrom about 0.75 to 300 mg, conveniently administered once, or in divideddoses 2 to 4 times, daily or in sustained release form. Unit dosageforms for oral administration thus for example may comprise from about0.2 to 75 or 150 mg or 300 mg, e.g. from about 0.2 or 2.0 to 10, 25, 50,75, 100, 150, 200 or 300 mg of the compound disclosed herein, togetherwith a pharmaceutically acceptable diluent or carrier therefor.

Pharmaceutical compositions comprising Compounds of the Invention may beprepared using conventional diluents or excipients and techniques knownin the galenic art. Thus oral dosage forms may include tablets,capsules, solutions, suspensions and the like.

Methods of Making Compounds of the Invention

The compounds of the Invention and their pharmaceutically acceptablesalts may be made using the methods as described and exemplified hereinand/or by methods similar thereto and/or by methods known in thechemical art. Such methods include, but not limited to, those describedbelow. If not commercially available, starting materials for theseprocesses may be made by procedures, which are selected from thechemical art using techniques which are similar or analogous to thesynthesis of known compounds.

Various NEP inhibitors and starting materials therefor may be preparedusing methods described in US 2006-0041014 A1, EP 1097719 A1, U.S. Pat.No. 8,513,244, and US 2013-0330365 A1. All references cited herein arehereby incorporated by reference in their entirety.

Various starting materials and/or Compounds of the Invention may beprepared using methods described in WO 2006/133261 and WO 2009/075784.All references cited herein are hereby incorporated by reference intheir entirety.

Terms and Abbreviations

-   -   DMF=N,N-dimethylforamide,    -   MeOH=methanol,    -   THF=tetrahydrofuran,    -   equiv.=equivalent(s),    -   h=hour(s),    -   HPLC=high performance liquid chromatography,    -   LiHMDS=lithium bis(trimethylsilyl)amide,    -   NMP=1-methyl-2-pyrrolidinone,    -   Pd₂(dba)₃=tris[dibenzylideneacetone]dipalladium(0),    -   TFA=trifluoroacetic acid,    -   TFMSA=trifluoromethanesulfonic acid    -   XantPhos=4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

EXAMPLES Example 1:3-((4-fluorophenyl)amino)-5,7,7-trimethyl-2-(pyridin-4-ylmethyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one

(a)2-(4-Bromobenzyl)-7-(4-methoxybenzyl)-5-methyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

A suspension of7-(4-methoxybenzyl)-5-methyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione(161 g, 562 mmol), 1-bromo-4-(bromomethyl) benzene (157 g, 628 mmol) andK₂CO₃ (93.2 g, 674 mmol) in DMF (800 mL) is stirred at room temperatureuntil the reaction is complete. The reaction mixture is poured intowater (5 L). After filtration, the filter cake is washed with water andethanol successively, and then dried under vacuum to give 226 g ofproduct (yield: 88%). MS (ESI) m/z 455.1 [M+H]⁺.

(b)2-(4-Bromobenzyl)-5-methyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

TFA (500 mL) is slowly added into a suspension of2-(4-bromobenzyl)-7-(4-methoxybenzyl)-5-methyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione(226 g, 496 mmol) in methylene chloride (320 mL), and then TFMSA (160mL) is added slowly. The reaction mixture is stirred at room temperatureovernight. Solvents are removed under reduced pressure. The obtainedresidue is treated with water (4 L) and ethyl acetate (2 L), stirred atroom temperature for 30 min, and then filtered. The filter cake isthoroughly washed with water to remove residual acids, followed bywashing with ethyl acetate. The obtained white solids are dried in aheated oven to give 159 g of product (yield: 96%). MS (ESI) m/z 335.0[M+H]⁺.

(c)6-Chloro-5-methyl-2-(4-bromobenzyl)-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one

2-(4-Bromobenzyl)-5-methyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione(159 g, 475 mmol) is suspended in POCl₃ (300 mL), and then slowly heatedto reflux. After the mixture is refluxed for 60 h, POCl₃ is removedunder reduced pressure. The obtained residue is dissolved in methylenechloride (5 L), cooled to 0° C., and then adjusted to pH 8-9 withsaturated sodium bicarbonate. After filtration, the obtained solids arewashed with water twice, and then dried under vacuum to give 157 g ofproduct (yield: 94%). MS (ESI) m/z 353.0 [M+H]⁺.

(d)6-(1-Hydroxy-2-methylpropan-2-ylamino)-5-methyl-2-(4-bromobenzyl)-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one

A mixture of6-chloro-5-methyl-2-(4-bromobenzyl)-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one(157 g, 444 mmol) and 2-amino-2-methylpropan-1-ol (236 g, 2.65 mol) inNMP (1.3 L) is heated at 120-125° C. for 2 h, and then poured into coldwater. After filtration, the filter cake is washed with water twice, andthen dried under vacuum to give 134 g of product (yield: 74%). MS (ESI)m/z 406.1 [M+H]⁺.

(e)2-(4-Bromobenzyl)-7,8-dihydro-5,7,7-trimethyl-[2H]-imidazo-[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one

Thionyl chloride (67 mL, 922 mmol) is added dropwise to a solution of6-(1-hydroxy-2-methylpropan-2-ylamino)-5-methyl-2-(4-bromobenzyl)-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one(134 g, 330 mmol) in DMF (800 mL). The reaction mixture is stirred atroom temperature until the reaction is complete. The mixture is pouredinto cold water, and then adjusted to pH 8-9 with ammonium hydroxideaqueous solution. After filtration, the obtained solids are washed withwater, and then dried under vacuum to give 118 g of product (yield:92%). MS (ESI) 388.1 [M+H]⁺.

(f)2-(4-Phenoxybenzyl)-7,8-dihydro-5,7,7-trimethyl-[2H]-imidazo-[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one

2-(4-Bromobenzyl)-7,8-dihydro-5,7,7-trimethyl-[2H]-imidazo-[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one(118 g, 304 mmol) is added into a suspension of phenol (57 g, 606 mmol)and cesium carbonate (200 g, 614 mmol) in NMP (900 mL), followed by2,2,6,6-tetramethylheptane-3,5-dione (7 mL, 33.5 mmol) and CuCl (15 g,152 mmol). The reaction mixture is heated at 120° C. under argonatmosphere for 10 h. After the completion of the reaction, the mixtureis diluted with water (4 L), and then extracted with ethyl acetate. Thecombined organic phase is evaporated to dryness. The obtained crudeproduct is purified by silica gel column chromatography to give 103 g ofproduct (yield: 84%). MS (ESI) m/z 402.2 [M+H]⁺.

(g)7,8-Dihydro-5,7,7-trimethyl-[2H]-imidazo-[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one

TFA (600 mL) is added into a suspension of2-(4-phenoxybenzyl)-7,8-dihydro-5,7,7-trimethyl-[2H]-imidazo-[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one(103 g, 257 mmol) in methylene chloride (210 mL) to give a tan solution,and then TFMSA (168 mL) is added. The reaction mixture is stirred atroom temperature until the starting material disappeared. The mixture ispoured into cold water (3 L). After filtration, the filter cake iswashed with water twice, and then basified with ammonium hydroxideaqueous solution, followed by adding ethyl acetate with stirring. Theprecipitated solids are filtered, washed successively with water threetimes, ethyl acetate twice and methanol once, and then dried undervacuum to give 45 g of product (yield: 80%). MS (ESI) m/z 220.2 [M+H]⁺.

(h)7,8-Dihydro-2-(4-methoxyphenyl)-5,7,7-trimethyl-[2H]-imidazo-[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one

A suspension of7,8-dihydro-5,7,7-trimethyl-[2H]-imidazo-[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one(3 g, 13.68 mmol), 1-(chloromethyl)-4-methoxybenzene (1.99 mL, 14.37mmol) and K₂CO₃ (3.78 g, 27.37 mmol) in DMF (50 mL) is stirred at roomtemperature until the reaction is complete. The solvent is removed, andthe residue is washed with water three times (3×70 mL), and then driedunder vacuum to give 4.3 g of crude product (yield: 93%), which is usedin the next step without further purification. MS (ESI) m/z 340.2[M+H]⁺.

(i)7,8-Dihydro-2-(4-methoxyphenyl)-3-chloro-5,7,7-trimethyl-[2H]-imidazo-[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one

1.0M LiHMDS (20 mL, 20 mmol) in THF is added dropwise into a solution of7,8-dihydro-2-(4-methoxyphenyl)-5,7,7-trimethyl-[2H]-imidazo-[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one(4.3 g, 12.67 mmol) and hexachloroethane (3.33 g, 13.94 mmol) inmethylene chloride (100 mL) at 25° C. The reaction mixture is stirred at25° C. for 30 min, and then quenched with water (150 mL). The methylenechloride phase is separated, and the water phase is extracted withmethylene chloride twice (2×60 mL). The combined methylene chloridephase is washed with brine three times (3×70 mL) and the methylenechloride is evaporated to dryness to give 4.6 g of crude product (yield:97%), which is used in the next step without further purification. MS(ESI) m/z 374.1 [M+H]⁺.

(j)7,8-Dihydro-3-chloro-5,7,7-trimethyl-[2H]-imidazo-[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one

To a suspension of7,8-dihydro-2-(4-methoxyphenyl)-3-chloro-5,7,7-trimethyl-[2H]-imidazo-[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one(4.6 g, 12.31 mmol) in TFA (23 mL) is added TFMSA (1.7 mL) slowly. Thereaction mixture is stirred at room temperature overnight. Solvents areremoved under reduced pressure. The obtained residue is treated withwater (100 mL), and then adjusted to pH 8-9 with 28% ammonium hydroxide(10 mL) at 0° C. After filtration, the filtrate is concentrated toapproximately 15 mL, and then extracted with 10% methanol in methylenechloride (5×60 mL). The combined organic phase is evaporated to drynessto give 1.94 g of crude product (yield: 62%), which is used in the nextstep without further purification. MS (ESI) m/z 254.1 [M+H]⁺.

(k)7,8-Dihydro-2-(pyridin-4-yl-methyl)-3-chloro-5,7,7-trimethyl-[2H]-imidazo-[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one

A suspension of7,8-dihydro-3-chloro-5,7,7-trimethyl-[2H]-imidazo-[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one(200 mg, 0.788 mmol), 4-chloromethyl pyridine (151 mg, 0.946 mmol) andK₂CO₃ (545 mg, 3.94 mmol) in DMF (7 mL) is stirred at 95° C. until thereaction is complete (approximately 2 h). The solvent is removed and theobtained crude product is purified on a 24 g neutral aluminum oxidecartridge, eluted with a gradient of 0 to 100% ethyl acetate in hexanesover 15 minutes, then eluted with ethyl acetate at a flow rate of 25ml/min for 30 minutes to give 128 mg of product (yield: 47%). MS (ESI)m/z 345.1 [M+H]⁺.

(l)7,8-Dihydro-2-(pyridin-4-yl-methyl)-3-(4-fluorophenylamino)-5,7,7-trimethyl-[2H]-imidazo-[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one

A suspension of7,8-dihydro-2-(pyridin-4-yl-methyl)-3-chloro-5,7,7-trimethyl-[2H]-imidazo-[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one(120 mg, 0.348 mmol), 4-fluoro-benzenamine (36 μL, 0.383 mmol) andpotassium carbonate (96 mg, 0.696 mmol) in tert-amyl alcohol (2.3 mL) isdegassed with argon and then Xantphos (8.1 mg, 0.0139 mmol) andPd₂(dba)₃ (6.4 mg, 0.00696 mmol) are added. The suspension is degassedagain, and then heated to 110° C. The reaction mixture is stirred at110° C. under argon for 8 h. After cooling to room temperature, themixture is filtered. The filtrate is evaporated to dryness under reducedpressure. The obtained residue is treated with DMF and then filteredwith a microfilter. The filtrate is purified with a semi-preparativeHPLC system using a gradient of 0-20% acetonitrile in water with 0.1%formic acid over 16 minutes to give 84 mg of the final product as aformate salt (HPLC purity: 98%; yield: 52%). 1H NMR (500 MHz,Chloroform-d) δ 8.50 (d, J=5.0 Hz, 2H), 8.18 (s, 1H), 7.26 (s, 1H),6.97-6.93 (m, 2H), 6.90-6.76 (m, 4H), 4.84 (s, 2H), 3.72 (s, 2H), 3.37(s, 3H), 1.45 (s, 6H). MS (ESI) m/z 420.2 [M+H]⁺.

Example 2:3-((4-fluorophenyl)amino)-5,7,7-trimethyl-2-((2-methylpyrimidin-5-yl)methyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one

(a)7,8-Dihydro-2-(2-methyl-pyrimidin-5-yl-methyl)-5,7,7-trimethyl-[2H]-imidazo-[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one

A suspension of7,8-Dihydro-5,7,7-trimethyl-[2H]-imidazo-[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one(200 mg, 0.92 mmol), 5-(chloromethyl)-2-methylpyrimidine (136 mg, 0.84mmol) and K₂CO₃ (350 mg, 2.56 mmol) in DMF (6 mL) is stirred at roomtemperature until the reaction is complete. The reaction mixture isfiltered, and the filter cake is rinsed with DMF. The collected filtrateis evaporated to dryness to give 242 mg of crude product (crude yield:78%), which is used in the next step without further purification. MS(ESI) m/z 326.2 [M+H]⁺.

(b)7,8-Dihydro-2-(2-methyl-pyrimidin-5-yl-methyl)-3-chloro-5,7,7-trimethyl[2H]-imidazo-[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one

1.0M LiHMDS (1.14 mL, 1.14 mmol) in THF is added dropwise into asolution of7,8-dihydro-2-(2-methyl-pyrimidin-5-yl-methyl)-5,7,7-trimethyl-[2H]-imidazo-[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one(370 mg, 1.14 mmol) and hexachloroethane (807 mg, 3.42 mmol) inmethylene chloride (4 mL) at 25° C. The reaction mixture is stirred atroom temperature for 30 min, and then diluted with methylene chloride(100 mL). The mixture is washed successively with saturated NaHCO₃solution (15 mL) and brine (15 mL), and then evaporated to dryness. Theobtained crude product is purified on a 24 g basic aluminum oxidecartridge, eluted with ethyl acetate at a flow rate of 25 mL/min to give402 mg of product (yield: 95%). MS (ESI) m/z 360.1 [M+H]⁺.

(c)7,8-Dihydro-2-(2-methyl-pyrimidin-5-yl-methyl)-3-(4-fluorophenylamino)-5,7,7-trimethyl-[2H]-imidazo-[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one

A suspension of7,8-dihydro-2-(2-methyl-pyrimidin-5-yl-methyl)-3-chloro-5,7,7-trimethyl-[2H]-imidazo-[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one(350 mg, 0.973 mmol), 4-fluorobenzenamine (110 μL, 1.167 mmol) andpotassium carbonate (269 mg, 1.946 mmol) in tert-amyl alcohol (3.0 mL)is degassed with argon and then Xantphos (45 mg, 0.0778 mmol) andPd₂(dba)₃ (36 mg, 0.039 mmol) are added. The suspension is degassedagain, and then heated to 110° C. The reaction mixture is stirred at110° C. under argon for 24 h. After cooling to room temperature, themixture is filtered. The filtrate is evaporated to dryness under reducedpressure. The obtained residue is treated with DMF and then filteredwith a microfilter. The filtrate is purified with a semi-preparativeHPLC system using a gradient of 0-20% acetonitrile in water with 0.1%formic acid over 16 minutes to give 356 mg of the final product as aformate salt (HPLC purity: 97%; yield: 74%). 1H NMR (500 MHz,Chloroform-d) δ 8.34 (s, 2H), 7.17 (s, 1H), 7.05-7.01 (m, 2H), 6.97-6.94(m, 2H), 4.84 (s, 2H), 3.75 (s, 2H), 3.37 (s, 3H), 2.70 (s, 3H), 1.48(s, 6H). MS (ESI) m/z 435.2 [M+H]⁺.

Example 3:3-((4-fluorophenyl)amino)-5,7,7-trimethyl-2-(pyridin-3-ylmethyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one

The synthesis method is analogous to Example 2 wherein 3-chloromethylpyridine is added in step (a) instead of5-(chloromethyl)-2-methylpyrimidine. The final product is obtained as aformate salt (HPLC purity: 96%; yield: 43%). 1H NMR (500 MHz,Chloroform-d) δ 8.52-8.5 (m, 2H), 8.23 (s, 1H), 7.35 (d, J=5.0 Hz, 1H),7.22-7.19 (m, 1H), 7.01-6.936.98 (m, 2H), 6.94-6.91 (m, 2H), 4.89 (s,2H), 3.78 (s, 2H), 3.42 (s, 3H), 1.50 (s, 6H). MS (ESI) m/z 420.2[M+H]⁺.

Example 4:(6aR,9aS)-2-(4-(6-hydroxypyridin-2-yl)benzyl)-5-methyl-3-(phenylamino)-5,6a,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one

(6aR,9aS)-2-(4-(6-fluoropyridin-2-yl)benzyl)-5-methyl-3-(phenylamino)-5,6a,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4-(2H)-one(20 mg, 0.039 mmol) is placed in a glass melting point tube and thenheated at 240° C. for 90 min. After cooled to room temperature, thematerial is dissolved in DMF and then purified with a semi-preparativeHPLC system using a gradient of 0-24% acetonitrile in water with 0.1%formic acid over 16 minutes to give 12 mg of the title compound as awhite solid (HPLC purity: 99%; yield: 60%). ¹H NMR (400 MHz,Chloroform-d) δ 10.47 (s, 1H), 7.49-7.35 (m, 3H), 7.28-7.16 (m, 2H),7.09-6.97 (m, 3H), 6.95 (s, 1H), 6.87 (d, J=7.9 Hz, 2H), 6.46 (d, J=9.1Hz, 1H), 6.35 (d, J=6.9 Hz, 1H), 4.89 (s, 2H), 4.78-4.61 (m, 2H), 3.32(s, 3H), 2.21 (dd, J=12.8, 6.1 Hz, 1H), 2.07-1.90 (m, 1H), 1.86-1.43 (m,4H). MS (ESI) m/z 506.2 [M+H]⁺.

Example 5:4-(((6aR,9aS)-5-methyl-4-oxo-3-(phenylamino)-4,5,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-2(6aH)-yl)methyl)benzenesulfonamide

A suspension of(6aR,9aS)-5-methyl-3-(phenylamino)-5,6a,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4-(2H)-one(100 mg, 0.31 mmol), 4-(bromomethyl)benzenesulfonamide (93 mg, 0.37mmol) and K₂CO₃ (52 mg, 0.37 mmol) in DMF (3 mL) is stirred at roomtemperature until the reaction is complete. The reaction mixture isfiltered and the collected filtrate is purified with a semi-preparativeHPLC system using a gradient of 0-35% acetonitrile in water with 0.1%formic acid over 16 minutes to give the title compound as an off-whitesolid (33 mg; HPLC purity: 97%; yield: 22%). ¹H NMR (500 MHz, CDCl₃) δ7.80 (d, J=8.0 Hz, 2H), 7.32-7.26 (m, 2H), 7.16-7.11 (m, 1H), 7.08 (d,J=8.0 Hz, 2H), 7.02 (s, 1H), 6.93 (d, J=7.9 Hz, 2H), 5.01-4.89 (m, 2H),4.84 (br, 2H), 4.83-4.76 (m, 2H), 3.37 (s, 3H), 2.28 (dd, J=12.5, 6.1Hz, 1H), 2.18-1.72 (m, 4H), 1.68-1.55 (m, 1H). MS (ESI) m/z 492.2[M+H]⁺.

Example 6:3-((4-fluorophenyl)amino)-5,7,7-trimethyl-2-((6-methylpyridin-3-yl)methyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one

(a)7-(4-methoxybenzyl)-5-methyl-2-((6-methylpyridin-3-yl)methyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

A suspension of7-(4-methoxybenzyl)-5-methyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione(6.28 g, 22 mmol), 5-(chloromethyl)-2-methylpyridine (3.1 g, 22 mmol)and K₂CO₃ (15 g, 110 mmol) in DMF (30 mL) and CH₂Cl₂ (30 mL) is stirredat room temperature until the reaction is complete. The reaction mixtureis diluted with water (300 mL) and then extracted with CH₂Cl₂ (60 mL×3).The combined organic phase is washed with 5% citric acid (40 mL×3) andbrine (40 mL×2), and then evaporated to dryness under reduced pressureto give 9.5 g of product as a pale yellow solid. MS (ESI) m/z 392.2[M+H]⁺.

(b)3-Chloro-7-(4-methoxybenzyl)-5-methyl-2-((6-methylpyridin-3-yl)methyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

1.0M LiHMDS (8 mL, 8 mmol) in THF is added dropwise into a solution of7-(4-methoxybenzyl)-5-methyl-2-((6-methylpyridin-3-yl)methyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione(783 mg, 2.0 mmol) and hexachloroethane (495 mg, 2.1 mmol) in methylenechloride (5 mL) at room temperature. The reaction mixture is stirred atroom temperature for an hour, and then quenched with water (100 mL). Themethylene chloride phase is separated, and the water phase is extractedwith methylene chloride (20 mL×3). The combined methylene chloride phaseis evaporated to dryness. The residue is purified on a silica columnusing a gradient of 0% to 20% methanol in ethyl acetate to give product(370 mg, 43% yield). MS (ESI) m/z 426.1 [M+H]⁺.

(c)3-(4-Fluorophenylamino)-7-(4-methoxybenzyl)-5-methyl-2-((6-methylpyridin-3-yl)methyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

A suspension of3-chloro-7-(4-methoxybenzyl)-5-methyl-2-((6-methylpyridin-3-yl)methyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione(210 mg, 0.49 mmol), 4-fluoro-benzenamine (108 mg, 0.97 mmol) andpotassium carbonate (202 mg, 1.46 mmol) in tert-amyl alcohol (2.5 mL) isdegassed with argon and then Xantphos (14 mg, 0.024 mmol) and Pd₂(dba)₃(12 mg, 0.013 mmol) are added. The suspension is degassed again, andthen heated to 110° C. The reaction mixture is stirred at 110° C. underargon for 24 h. After cooled to room temperature, the mixture is dilutedwith water (100 mL) and then extracted with CH₂Cl₂ (30 mL×3). Thecombined organic phase is evaporated to dryness under reduced pressureto give 250 mg of crude product, which is used in the next step withoutfurther purification. MS (ESI) m/z 501.3 [M+H]⁺.

(d)3-(4-Fluorophenylamino)-5-methyl-2-((6-methylpyridin-3-yl)methyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

TFA (1 mL) is slowly added into a suspension of crude3-(4-fluorophenylamino)-7-(4-methoxybenzyl)-5-methyl-2-((6-methylpyridin-3-yl)methyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione(250 mg) in methylene chloride (6 mL), and then TFMSA (0.5 mL) is addedslowly. The reaction mixture is stirred at room temperature for an hour.Solvents are removed under reduced pressure. The residue is dissolved inCH₂Cl₂ (10 mL) and then slowly added into a cold saturated NaHCO₃aqueous solution (30 mL), followed by extraction with CH₂Cl₂ (15 mL×3).The combined organic phase is evaporated to dryness under reducedpressure and further dried on a vacuum pump to give 300 mg of crudeproduct, which is used in the next step without further purification. MS(ESI) m/z 381.1 [M+H]⁺.

(e)6-chloro-3-(4-fluorophenylamino)-5-methyl-2-((6-methylpyridin-3-yl)methyl)-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one

Crude3-(4-fluorophenylamino)-5-methyl-2-((6-methylpyridin-3-yl)methyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione(180 mg) is suspended in POCl₃ (10 mL) and then heated at 180° C. in amicrowave reactor for an hour. After the mixture is cooled to roomtemperature, POCl₃ is removed under reduced pressure. The obtainedresidue is washed with hexanes and then dissolved in CH₂Cl₂:MeOH (10:1).The solution is washed with saturated NaHCO₃ aqueous solution and thenextracted with CH₂Cl₂: MeOH (10:1) three time. The combined organicphase is evaporated to dryness under reduced pressure and then furtherdried under vacuum to give 120 mg of crude product as a grey solid,which is used in the next step without further purification. MS (ESI)m/z 399.1 [M+H]⁺.

(f)3-(4-Fluorophenylamino)-6-(1-hydroxy-2-methylpropan-2-ylamino)-5-methyl-2-((6-methylpyridin-3-yl)methyl)-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one

A mixture of crude6-chloro-3-(4-fluorophenylamino)-5-methyl-2-((6-methylpyridin-3-yl)methyl)-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one(120 mg), DIPEA (0.6 mL) and 2-amino-2-methylpropan-1-ol (300 mg, 3.4mmol) in DMA (10 mL) in a sealed vial is heated at 105° C. for 20 h.After cooled to room temperature, the mixture is diluted with saturatedNaHCO₃ aqueous solution (100 mL) and then extracted with CH₂Cl₂ (30mL×3). The combined organic phase is evaporated to dryness under reducedpressure. The residue is purified with a semi-preparative HPLC systemusing a gradient of 0-20% acetonitrile in water containing 0.1% formicacid over 16 min to give 26 mg of product. MS (ESI) m/z 452.2 [M+H]⁺.

(g)3-((4-Fluorophenyl)amino)-5,7,7-trimethyl-2-((6-methylpyridin-3-yl)methyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one

Thionyl chloride (two drops) is added dropwise to a solution of3-(4-fluorophenylamino)-6-(1-hydroxy-2-methylpropan-2-ylamino)-5-methyl-2-((6-methylpyridin-3-yl)methyl)-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one(26 mg, 0.058 mmol) in DMF (0.5 mL) and CH₂Cl₂ (1 mL). The reactionmixture is stirred at room temperature for 30 min. After the solventsare removed under reduced pressure, the residue is purified with asemi-preparative HPLC system using a gradient of 0-20% acetonitrile inwater containing 0.1% formic acid over 16 min to give 13.9 mg of productas an off-white solid (56% yield; HPLC purity: 95%). ¹H NMR (500 MHz,Chloroform-d) δ 8.12 (s, 1H), 7.29-7.25 (m, 1H), 7.06 (d, J=7.9 Hz, 1H),7.00 (t, J=8.4 Hz, 2H), 6.97-6.89 (m, 2H), 4.83 (s, 2H), 3.74 (s, 2H),3.37 (s, 3H), 2.52 (s, 3H), 1.47 (s, 6H). MS (ESI) m/z 434.2 [M+H]⁺.

Example 7:4-((3-((4-fluorophenyl)amino)-5,7,7-trimethyl-4-oxo-4,5,7,8-tetrahydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-2-yl)methyl)benzenesulfonamide

A suspension of3-((4-fluorophenyl)amino)-5,7,7-trimethyl-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one(132 mg, 0.50 mmol), 4-(bromomethyl)benzenesulfonamide (101 mg, 0.40mmol) and Na₂CO₃ (43 mg, 0.41 mmol) in DMF (4 mL) is stirred at roomtemperature overnight. The reaction mixture is filtered and thecollected filtrate is purified with a semi-preparative HPLC system usinga gradient of 0-20% acetonitrile in water with 1% formic acid over 16minutes to give the title compound as an off-white solid (8.8 mg; HPLCpurity: 97%). ¹H NMR (500 MHz, Chloroform-d) δ 7.81 (d, J=8.2 Hz, 2H),7.09 (d, J=8.2 Hz, 2H), 6.97 (t, J=8.4 Hz, 2H), 6.93-6.86 (m, 3H), 4.88(s, 2H), 4.79 (br, 2H), 3.71 (s, 2H), 3.36 (s, 3H), 1.43 (s, 6H). MS(ESI) m/z 498.1 [M+H]⁺.

Example 8:3-((4-fluorophenyl)amino)-2-(4-(6-hydroxypyridin-2-yl)benzyl)-5,7,7-trimethyl-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one

3-((4-fluorophenyl)amino)-2-(4-(6-fluoropyridin-2-yl)benzyl)-5,7,7-trimethyl-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one(350 mg, 0.68 mmol) is suspended in water (3 mL), followed by addingconcentrated HCl (3 mL) and dioxane (6 mL). The mixture is heated atreflux for 24 h. After the solvents are removed under reduced pressure,the residue is purified with a semi-preparative HPLC system using agradient of 0-22% acetonitrile in water with 1% formic acid over 16minutes to give 240 mg of the title compound as an off-white solid (HPLCpurity: 95%; yield: 69%). ¹H NMR (400 MHz, Chloroform-d) δ 7.59-7.46 (m,3H), 7.10 (d, J=8.3 Hz, 2H), 7.02-6.85 (m, 4H), 6.55 (dd, J=9.1, 1.0 Hz,1H), 6.47 (dd, J=7.0, 1.0 Hz, 1H), 4.96 (s, 2H), 3.79 (s, 2H), 3.37 (s,3H), 1.50 (s, 6H). MS (ESI) m/z 512.2 [M+H]⁺.

Example 9:2-(4-(6-hydroxypyridin-2-yl)benzyl)-3,5,7,7-tetramethyl-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one

The synthesis method is analogous to Example 8 wherein2-(4-(6-fluoropyridin-2-yl)benzyl)-3,5,7,7-tetramethyl-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-oneis used as the starting material instead of3-((4-fluorophenyl)amino)-2-(4-(6-fluoropyridin-2-yl)benzyl)-5,7,7-trimethyl-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one.Final product is obtained as a white solid (HPLC purity: 98%; yield:65%). ¹H NMR (500 MHz, Chloroform-d) δ 8.13 (s, 1H), 7.65 (d, J=8.3 Hz,2H), 7.57 (dd, J=9.1, 7.0 Hz, 1H), 7.29 (d, J=8.3 Hz, 2H), 6.61 (dd,J=9.2, 1.0 Hz, 1H), 6.56 (dd, J=7.1, 1.0 Hz, 1H), 5.28 (s, 2H), 3.82 (s,2H), 3.50 (s, 3H), 2.57 (s, 3H), 1.51 (s, 6H). MS (ESI) m/z 417 [M+H]⁺.

Example 10:3-ethyl-2-(4-(6-hydroxypyridin-2-yl)benzyl)-5,7,7-trimethyl-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one

A solution of3-ethyl-2-(4-(6-fluoropyridin-2-yl)benzyl)-5,7,7-trimethyl-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one(865 mg, 2.0 mmol) in 1N HCl (3 mL) in a sealed microwave vial is heatedin a microwave reactor at 100° C. for an hour. After cooled to roomtemperature, the mixture is neutralized to pH 5-6 with 1N NaOH, dilutedwith 9 mL of DMF, and then filtered. The filtrate is purified with asemipreparative HPLC system using a gradient of 0-19% acetonitrile inwater with 1% formic acid over 16 minutes to give 150 mg of the titlecompound as an off-white solid (HPLC purity: 99%; yield: 17%). MS (ESI)m/z 431 [M+H]⁺.

Example 11: Measurement of PDEI Inhibition In Vitro Using IMAPPhosphodiesterase Assay Kit

Phosphodiesterase 1 (including PDE1A, PDE1B and PDE1C) is acalcium/calmodulin dependent phosphodiesterase enzyme that convertscyclic guanosine monophosphate (cGMP) to 5′-guanosine monophosphate(5′-GMP). PDEIB can also convert a modified cGMP substrate, such as thefluorescent molecule cGMP-fluorescein, to the correspondingGMP-fluorescein. The generation of GMP-fluorescein from cGMP-fluoresceincan be quantitated, using, for example, the IMAP (Molecular Devices,Sunnyvale, Calif.) immobilized-metal affinity particle reagent.

Briefly, the IMAP reagent binds with high affinity to the free5′-phosphate that is found in GMP-fluorescein and not incGMP-fluorescein. The resulting GMP-fluorescein-IMAP complex is largerelative to cGMP-fluorescein. Small fluorophores that are bound up in alarge, slowly tumbling, complex can be distinguished from unboundfluorophores, because the photons emitted as they fluoresce retain thesame polarity as the photons used to excite the fluorescence.

In the phosphodiesterase assay, cGMP-fluorescein, which cannot be boundto IMAP, and therefore retains little fluorescence polarization, isconverted to GMP-fluorescein, which, when bound to IMAP, yields a largeincrease in fluorescence polarization (Amp). Inhibition ofphosphodiesterase, therefore, is detected as a decrease in Amp.

Enzyme Assay

Materials: All chemicals are available from Sigma-Aldrich (St. Louis,Mo.) except for IMAP reagents (reaction buffer, binding buffer, FL-GMPand IMAP beads), which are available from Molecular Devices (Sunnyvale,Calif.).

Assay: Phosphodiesterase enzymes that may be used include:3′,5′-cyclic-nucleotide-specific bovine brain phosphodiesterase (Sigma,St. Louis, Mo.) (predominantly PDEIB but also contains PDE1A and PDE1C)and recombinant full length human PDE1A, PDE1B and PDE1C which may beproduced e.g., in HEK or SF9 cells by one skilled in the art. The PDE1enzyme is reconstituted with 50% glycerol to 2.5 U/ml. One unit ofenzyme will hydrolyze 1.0 μmol of 3′,5′-cAMP to 5′-AMP per min at pH 7.5at 30° C. One part enzyme is added to 1999 parts reaction buffer (30 μMCaCl₂, 10 U/ml of calmodulin (Sigma P2277), 10 mM Tris-HCl pH 7.2, 10 mMMgCl₂, 0.1% BSA, 0.05% NaN₃) to yield a final concentration of 1.25mU/ml. 99 μl of diluted enzyme solution is added into each well in aflat bottom 96-well polystyrene plate to which 1 μl of test compounddissolved in 100% DMSO is added. The compounds are mixed andpre-incubated with the enzyme for 10 min at room temperature. The FL-GMPconversion reaction is initiated by combining 4 parts enzyme andinhibitor mix with 1 part substrate solution (0.225 μM) in a 384-wellmicrotiter plate. The reaction is incubated in dark at room temperaturefor 15 min. The reaction is halted by addition of 60 μl of bindingreagent (1:400 dilution of IMAP beads in binding buffer supplementedwith 1:1800 dilution of antifoam) to each well of the 384-well plate.The plate is incubated at room temperature for 1 hour to allow IMAPbinding to proceed to completion, and then placed in an Envisionmultimode microplate reader (PerkinElmer, Shelton, Conn.) to measure thefluorescence polarization (Amp).

A decrease in GMP concentration, measured as decreased Amp, isindicative of inhibition of PDE activity. IC₅₀ values are determined bymeasuring enzyme activity in the presence of 8 to 16 concentrations ofcompound ranging from 0.0037 nM to 80,000 nM and then plotting drugconcentration versus Amp, which allows IC₅₀ values to be estimated usingnonlinear regression software (XLFit; IDBS, Cambridge, Mass.).

The Compounds of the Invention are tested in an assay as described orsimilarly described herein for PDE1 inhibitory activity, which compoundsgenerally have IC₅₀ values against bovine PDE1 of less than 75 nM and/orPDE1B (HEK/Sf9) of less than 10 nM and/or PDE1A (HEK) and/or PDE1C (HEK)of less than 1 nM.

Example 12: Pharmacokinetic Analysis of the Compounds of the Invention

Animals: Male, C57BL/6 mice (Jackson Labs, 25-30 g in body weight) areprovided by Jackson Laboratories. Up to five mice are housed per cageand are maintained under a 12 hour light/dark cycle with access to foodand water ad libitum. All procedures for the handling and use of animalsfollow the guidelines of the Institutional Animal Care and Use Committee(IACUC) of Columbia University, in accordance with NIH guidelines. Eightweek-old mice (N=3/dose level or treatment group) are used in theexperiments.

Experimental Treatment: Compounds: Selected Compounds of the Inventionare evaluated in the present study. Formulation/Vehicle: 3% 1N HCl, 5%Labrasol and 92% of 5% TPGS in 0.05M Citrate buffer (CB, pH 4.0). Thetest compound(s) are prepared as solution in vehicle and are dosed in avolume of 8 ml/kg.

Compound Preparation: Powdered stocks of the test compound(s) aremeasured and dissolved in 3% 1N HCl, 5% Labrasol and 92% of 5% TPGS in0.05M Citrate buffer (CB, pH 4.0). Two or three layers of 3 mm glassbeads are added to the bottom of the 10 ml glass tube to promote mixing.The tube is mixed using a benchtop vortex mixer then sonicated using aVWR sonicator (model 750) for about 5 min until the drug powder istotally dissolved in into a vehicle solution.

Treatment of Animals: Mice (N=3 mice/dose/time point) receive a 10 mg/kgoral (PO) dose of the test compound(s) at time 0. Groups of mice arekilled at a specified time point, either 0.25, 0.5, 1, or 2 h after drugadministration. Brain tissue is collected and frozen at −80° C., untilanalysis. Blood is collected from the mice by puncture of theretro-orbital vein using a Pasteur pipette (VWR, Cat#53283-911), thendeposited into silicon-coated blood collection tubes containing 0.105Msodium citrate solution (BD Vacutainer, #366392, Franklin Lakes, N.J.).Blood samples are centrifuged at the speed of 8000 g for 40 minutes in4° C. (TOMY, refrigerated benchtop microcentrifuge, Fremont, Calif.94583) and plasma decanted into Eppendorf tubes and frozen at −80° C.until analysis. Plasma and brain tissue samples are processed andanalyzed by the analytical group using LC-MS/MS methods, as describedbelow.

Sample Preparation: Samples of plasma are prepared for analysis asfollows: 50 μL of the plasma samples is transferred into a 500 μlpolypropylene microtube (Eppendorf Cat#022363611) as follows:

Standards Samples 50 μL control (blank) plasma 50 μL test sample plasma10 μL standard working solution 10 μL 1:1 Methanol:Water in 1:1Methanol:Water 150 μL 0.1 μM Standard in 150 μL 0.1 μM Standard Methanolin Methanol

Each tube is vortex mixed, then centrifuged for 20 min at 15000 rpm. Thesupernatant is collected and 100 μL of each is then transferred into a96-well polypropylene Elisa plate for mass spectrometric analysis.

Samples of brain homogenate were prepared for analysis as follows:Approximately 0.5 g of brain tissue is weighed and homogenized with 1 mLMilli-Q water. Then 60 μL of the resulting homogenate is thentransferred into a clean 500 μL polypropylene microtube (EppendorfCat#022363611) and treated as shown below:

Standards Samples 60 μL control (blank) brain 60 μL test sample brainhomogenate homogenate 20 μL standard working solution in 20 μL 1:1Methanol:Water 1:1 Methanol:Water 180 μL 0.1 μM Standard in 180 μL 0.1μM Standard in Methanol Methanol

Each tube is vortexed, then centrifuged for 20 min at 15000 rpm using aTomy benchtop centrifuge at 4° C. Standard is an internal standard usedfor LC-MS/MS quantitation. 150 μL of each supernatant is thentransferred into a 96-well plate for mass spectrometric analysis. Anyremaining plasma or homogenate is stored at approximately −20° C.pending any necessary repeat analysis. For each test sample, acalibration curve is prepared covering the range of 0.5-500 ng/mL.

HPLC and Mass Spectrometric Analysis: Analysis to quantify theconcentration of each compound in plasma and brain homogenate is carriedout using reverse phase HPLC followed by mass spectrometric detectionusing the parameters listed:

HPLC: Waters Alliance 2795 HT Mobile phase A: 0.1% Formic acid in waterMobile phase B: 0.1% Formic acid in methanol Column: Phenomenex Synergi4μ Fusion-RP 50 × 2 mm Column Temperature: 40° C. Flow Time SolventSolvent Rate (min) A (%) B (%) (mL/min) 0 80   20 0.6 2 0 100 0.6 4 0100 0.6 Waters Alliance 2795 LC rapid equilibration flow 5 (mL/min):Waters Alliance 2795 LC rapid equilibration time 0.25 (min):Re-equilibration time (min): 1 Injection volume (μl): 10Each compound is detected and quantified using Multiple ReactionMonitoring (MRM) of positive electrospray mode with a WatersQuattroMicro™ mass spectrometry system.

RESULTS: Plasma and Brain Analysis: All the test compound(s) testedcould be detected and analyzed when spiked into control plasma. Standardcurves are established prior to the analysis of the samples and provedlinear over the range of 0.5-1500 ng/mL in plasma and 0.5-500 ng/mL inbrain. Plasma and brain levels of each compound are determined andexpressed as means±standard deviation for each compound at each timepoint. Brain and plasma C_(max) and T_(max) values are estimated foreach compound by visual inspection of the data. A ratio of brain/plasmaconcentration (B/P) is also calculated for each compound by dividingBrain AUC_((0-2h))/Plasma AUC_((0-2h)) or Brain C_(max)/Plasma C_(max).

Results: Using the procedure described or similarly described, thecompounds of Examples 2-6 and 8-10 are tested and display excellent oralbioavailability to the plasma with correspondingly low brain exposure(B/P ratios ranging from 0.02-0.33). In particular, the compound ofExamples 4-5 and 7-10 have a B/P ratio of less than about 0.05. Thecompound of Examples 1-3 have a B/P ratio of about 0.1 or less. Thecompound of Example 6 has a B/P ratio of about 0.3. In contrast, the B/Pratio of(6aR,9aS)-3-(phenylamino)-5,6a,7,8,9,9a-hexahydro-5-methyl-2-(4-(trifluoromethyl)-benzyl)-cyclopent[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-oneis about 4.5-4.8.

Example 13: Effects of PDE1 Inhibition on Isoproterenol Induced CardiacHypertrophy

The compound of Example 2 is tested in a mouse model where the mice aretreated with isoproterenol. Such a model can be useful for extrapolatingto diseases or disorders involving an enlargement of the heart orcardiac tissue, e.g., congestive heart disease.

Animals: 129S1/SvlmJ mice, 20-40 grams, Jackson Laboratories (BarHarbor, Me.).

Number and Sex: 40 males.

Housing and Enrichment: Animals are housed individually during the studyin shoe-box style cages in an environmentally controlled room.

Food: Animals are provided standard diet food ad libitum.

Water: Animals are provided tap water ad libitum.

Contaminants: There are no known contaminants in the food or water thatwould interfere with this study.

Environmental Conditions: The animal room temperatures are set tomaintain 18 to 24° C. A 12-hour dark cycle are implemented unlessinterrupted by study procedures.

Animal Selection: Animals are selected for the study based on healthstatus, body weight, or other relevant data as appropriate.

Test Compound of Example 2: Test compound of Example 2 is supplied as apowder. The test article dosing solutions are prepared on the day ofadministration by dissolving the test compound in the vehicle (0.9%sodium chloride for injection, U.S.P.) at concentration of 2.0 mg/mLstock/dosing solution. The compound are agitated with a vortex mixer andsonicated to ensure the compound is in solution. The 2.0 mg/mL solutionis diluted with vehicle to create the 0.2 mg/mL and the 0.6 mg/mL dosingsolutions. Dosing solutions are stored in a refrigerator between thedaily doses and are allowed to return to room temperature before theevening dose.

Test Compound Isoproterenol: Test compound isoproterenol is supplied bySigma (St. Louis, Mo.) as a powder. The test article dosing solutionsare prepared on the day of administration by dissolving the testcompound in the vehicle (0.9% sodium chloride for injection, U.S.P.) atconcentration of 2.0 mg/mL. The compound is agitated with a vortex mixerand sonicated to ensure the compound is in solution.

Groups 4 5 6 1 2 3 Exam- Exam- Exam- Test Article NA NA Vehicle ple 2ple 2 ple 2 Test Article NA NA 0   0.2   0.6 2 Concentration (mg/mL)Test Article NA NA 0 1 3 10  Dosage3 (mg/kg) Test Article NA NA 5 5 5 5Dose Volume (mL/kg) Isoproterenol 0 2 2 2 2 2 Concentration (mg/mL)Isoproterenol  0¹ 10¹ 10² 10² 10² 10² Dosage (mg/kg) Isoproterenol 5 5 55 5 5 Dose Volume (mL/kg) Number of 6 6 7 7 7 7 Male Mice Vehicle is0.9% sodium chloride for injection, U.S.P. ¹Mice will receive one bolussubcutaneous injection at the listed dose once/day from days 1-13. ²Micewill receive one bolus subcutaneous injection at the listed doseonce/day from days 4-9. 3Mice will receive one bolus intraperitonealinjection at listed dose twice/day from days 1-9.

Mice in group 1 and 2 are dosed daily via subcutaneous administration.

On days 7, 10 and 14, 2 animals from groups 1 and 2 are weighed and theneuthanized via CO2 asphyxiation. Hearts are harvested and gently infusedwith ice cold saline until clear. The entire heart is weighed. The atriaare removed and the left ventricle (with septum) is separated from theright ventricle. The ventricles are weighed separately. The left tibiais removed and separated from the soft tissue. Longitudinal measurementare obtained with a digital micrometer.

Mice in groups 3-6 are dosed once/day with isoproterenol subcutaneouslyfrom Days 4-9 and twice/day with vehicle or test compound viaintraperitoneal administration from Days 1 to 9.

Groups 3-6 Terminal Procedure

On Day 10 (based on the results from Groups 1 and 2) animals from eachgroup are weighed and then euthanized via CO2 asphyxiation. Hearts areharvested and gently infused with ice cold krebs-henseleit buffer (Sigma3753) until clear. The entire heart is weighed. The atria are removedand the left ventricle (with septum) is separated from the rightventricle. The ventricles are weighed separately. The ventricles arestored in ice cold krebs-henseleit buffer for approximately 15 minutesand then transferred to 10% buffered formalin at room temperature untilpossible shipment for possible future analysis.

The left tibia is removed and separated from the soft tissue.Longitudinal measurement is obtained with a digital micrometer.

The dissected atria and tibia are disposed of with the rest of thecarcass.

Prior to cardiectomy, a 0.5 mL sample of blood is withdrawn into acollection tube containing K2-EDTA anticoagulant. The sample is storedon ice until spun in a refrigerated centrifuge. Plasma is separated andfrozen on dry ice, and then stored in a freezer set to maintainapproximately −20° C. until shipped on dry ice for possible futureanalysis.

Using the procedure as described or similarly described above, theCompound of Example 2 of the invention is evaluated and provides thefollowing results:

HW/TL Ratio HW/TL Ratio Group (mean) (std error) Vehicle 9.2427 0.3364 1mg/kg Cmpd 2 8.4876 0.3507 3 mg/kg Cmpd 2 8.0830 0.1489 10 mg/kg Cmpd 28.0976 0.2279Isoproterenol treatment in mice increases cardiac size (Size isindicated by heart weight (g)/tibia length (mm)) in mice not treatedwith the compound of Example 2. Oral administration of up to 10 mg/kgresults in a statistically significant effect on the development ofisoproterenol-induced cardiac hypertrophy. Specifically, at 1 mg/kg,there is about an 8% reduction in the heart weight/tibia length (HW/TL)ratio compared to vehicle, while at both 3 mg/kg and at 10 mg/kg, thereis about a 12.5% reduction in HW/TL ratio compared to vehicle. Thecompound of Example 2 also significantly prevents cardiac hypertrophy ata dose of both 3 mg/kg and 10 mg/kg. Isoproteranol-induced cardiacenlargement, as measured by the (LV+RV)/TL ratio (left ventricle plusright ventricle weight/tibia length), is decreased by 10% compared tovehicle for both 3 mg/kg and 10 mg/kg dosing.

Example 14: Effects of PDE1 Inhibition on Cardiac Hypertrophy inTransverse Aortic Constriction (TAC) Mouse Model

Animals: C59Bl/6 mice, 20-40 grams, Charles River (Portage, Mich.).

Number and Sex: 35 males.

Housing and Enrichment: Animals are housed individually during the studyin shoe-box style cages in an environmentally controlled room.

Food: Animals are provided standard diet food ad libitum.

Water: Animals are provided tap water ad libitum.

Environmental Conditions: The animal room temperatures are set tomaintain 18 to 24° C. A 12-hour dark cycle is implemented unlessinterrupted by study procedures.

Animal Selection: Animals are selected for the study based on healthstatus, body weight, or other relevant data as appropriate.

Test Compound of Example 2: Test compound of Example 2 is formulated.The test article dosing solutions are prepared by titrating the freebase with 1 mole/mole dilute (0.02N) HCl and diluting the dissolved testcompound in the vehicle (0.5% methylcellulose in distilled water) to afinal concentration of 0.45 mg/mL and 1.5 mg/mL dosing solution. Asupply of formulation for thirty days of dosing are prepared (day 1-6for 1^(st) surgical cohort).

Reference Test Compound—Losartan potassium: Losartan is obtained byCorDynamics, and prepared on the day of administration by dissolving inthe vehicle (0.5% methylcellulose in distilled water) at concentrationof 4.5 mg/mL dosing solution. The compound is agitated with a vortexmixer and sonicated to ensure the compound is in solution.

Groups 1 2 3 4 5 Test Article Vehicle Vehicle Example 2 Example 2Losartan Test Article NA 0 0.45   1.5   4.5 Concentration (mg/mL) TestArticle NA  0¹ 3¹  10¹ 30² Dosage (mg/kg/dose) Test Article NA   6.676.67   6.67   6.67 Dose Volume (mL/kg/dose) Number of Male 7 7 7   7 7Mice Group 1 animals receive sham transverse aortic constriction andGroup 2-5 animals receive transverse aortic constriction at day −1.Vehicle is 0.5% methylcellulose. ¹Mice receive oral dose as listed abovetwice/day from day 1 post-surgery until day 28. ²Mice receive oral doseas listed above once/day from day 1 post-surgery until day 28

Transverse Aortic Constriction Surgery: Animals are anesthetized withisoflurane using a nose cone connected to a vaporizer that delivers 1-2%isoflurane driven by 100% oxygen. After initial anesthesia withsoflurane, mice receive 1 mg/kg Buprenex SR (1 mg/mL, s.c.) and 10 mg/kgof etomidate (2 mg/mL, i.p.). Animals are intubated for mechanicalventilation. The endotracheal tube is connected to a mechanicalventilator that provides positive-pressure ventilation with 1-2%isoflurane remainder oxygen, set to maintain ˜120 breaths/min with atidal volume of ˜200 μL. Body temperature is monitored throughout theexperiment.

A sternotomy is performed from the manubrium through the second or thirdrib in order to expose the aorta in the thoracic cavity. The aorta iscleaned of surrounding tissue and the transverse aorta clamped with atitanium microclip, the diameter similar to a 27G needle. The transverseaorta from mice in Group 1 is cleaned of surrounding tissue but notclamped. The incision is closed, endotracheal tube is removed and animalis allowed to recover.

Immediately following completion of the surgical procedure, mice isplaced in a recovery cage in the recovery room. The animals is returnedto general housing once alert and ambulatory.

Length of treatment post-operatively is determined by return to normalpre-operative function. This is judged by attitude, activity level, andappetite. Daily post-op monitoring is performed for 4 days post-op.Stitches are removed when the surgical sites have healed sufficiently,approximately 10-14 days after surgery.

Mice are dosed twice daily with vehicle or the compound of Example 2 viaoral administration with approximately 6 hours between each dosebeginning at 1 day post-surgery. Mice are dosed once daily with losartanvia oral administration. Body weights are taken after arrival, prior tosurgery, prior to dosing on the first day of dosing and every third dayof dosing for dose calculation purposes.

On day 28, echocardiographic measurements are performed for allexperimental groups. Animals are anesthetized with isoflurane using anose cone connected to a vaporizer that delivers 1-2% isoflurane drivenby 100% oxygen. Mice are placed in the dorsal decubitus position on awarming platform to maintain body temperature at 37±0.5° C. Heart rateis monitored throughout the experiment. Transthoracic two-dimensional,B-mode, M-mode and pulsed Doppler images are acquired with ahigh-resolution echocardiographic system (VeVo 770, Visual Sonics,Toronto, ON, Canada) equipped with a 30-MHz mechanical transducer.

After echocardiographic measurements, animals are euthanized via CO₂asphyxiation. Hearts and lungs are harvested and gently infused with icecold krebs-henseleit buffer (Sigma 3753) until clear. Lungs are weighedand the right and left lung are separated. The right and left lung areimmediately frozen in liquid nitrogen and placed into separate tubes.The entire heart is weighed. The atria are removed and the leftventricle (with septum) is separated from the right ventricle. Theventricles are weighed separately before being divided into two pieces.Ventricle pieces are frozen in liquid nitrogen and placed into separatetubes. Liquid nitrogen samples are stored in a freezer set to maintainapproximately −80° C. until shipped on dry ice to the Sponsor forpossible future analysis.

The left tibia is removed and separated from the soft tissue.Longitudinal measurement will be obtained with a digital micrometer. Thedissected tibia is disposed of with the rest of the carcass.

Prior to cardiectomy, a 0.5 mL sample of blood is withdrawn into acollection tube containing sodium citrate anticoagulant. The sample isstored on ice until spun in a refrigerated centrifuge. Plasma isseparated and frozen on dry ice, and then stored in a freezer set tomaintain approximately −20° C. until shipped on dry ice for possiblefuture analysis.

Using the procedures described or similarly described above, thefollowing results are obtained:

Terminal LV + S/TL Terminal LV + S/TL Group Ratio (mean) Ratio (stderror meas) Sham Operation 4.6 0.14 Vehicle 6.6 0.32 3 mg/kg Cmpd 2 5.80.25 10 mg/kg Cmpd 2 5.8 0.46 40 mg/kg Losartan 6.2 0.33The compound of Example 2 at 3 mg/kg and 10 mg/kg shows a tendency toreverse the cardiac hypertrophy induced by the TAC constriction.Hypertrophy is judged based on the heart size (left ventricle+septumweight in mg) normalized to animal size (tibia length in mm), defined asthe “TAC ratio”. Mice that receive the sham operation show a TAC ratioof 4.6 (mg/mm), while mice that receive the compound of Example 2 ateither 3 mg/kg or 10 mg/kg show a TAC ratio of 5.8. In contrast, micethat receive vehicle show a TAC ratio of 6.6, and mice that receiveLosartan show a TAC ratio of 6.2. The different between the sham andvehicle TAC groups is statistically significant, while the differencebetween the sham and compound of Example 2 groups is not statisticallysignificant. The difference between the vehicle group and the Losartangroup is also not statistically significant. Thus, the compound ofExample 2 shows a tendency to reduce TAC-induced hypertrophy.

What is claimed is:
 1. A compound of Formula I:

wherein (i) R₁ is C₁₋₄alkyl or —NH(R₂), wherein R₂ is phenyl optionallysubstituted with halo, for example, 4-fluorophenyl; (ii) X, Y and Z are,independently, N or C; (iii) R₃, R₄ and R₅ are independently H orC₁₋₄alkyl; or R₃ is H and R₄ and R₅ together form a tri-methylene bridge(pref. wherein the R₄ and R₅ together have the cis configuration, e.g.,where the carbons carrying R₄ and R₅ have the R and S configurations,respectively), (iv) R₆, R₇ and R₈ are independently: H, C₁₋₄alkyl,pyrid-2-yl substituted with hydroxy, or —S(O)₂—NH₂; (v) Provided thatwhen X, Y and/or Z are N, then R₆, R₇ and/or R₈, respectively, are notpresent; and when X, Y and Z are all C, then at least one of R₆, R₇ orR₈ is —S(O)₂—NH₂ or pyrid-2-yl substituted with hydroxy; in free or saltform.
 2. The compound according to claim 1, wherein R₃, R₄ and R₅ areindependently H or C₁₋₄alkyl, in free or salt form.
 3. The compoundaccording to claim 1, wherein R₃ and R₄ are independently C₁₋₄alkyl andR₅ is H, in free or salt form.
 4. The compound according to claim 1,wherein R₃ and R₄ are both methyl and R₅ is H, in free or salt form. 5.The compound according to claim 1, wherein R₁ is —NH(R₂), wherein R₂ isphenyl optionally substituted with halo, in free or salt form.
 6. Thecompound according to claim 1, wherein R₁ is —NH(R₂), wherein R₂ isphenyl substituted with halo, for example, 4-fluorophenyl, in free orsalt form.
 7. The compound according to claim 1, wherein at least one ofX, Y and/or Z is N, the corresponding R₆, R₇ and/or R₈ do not exist, andthe remaining R₆, R₇ and/or R₈ are independently H or C₁₋₄alkyl, in freeor salt form.
 8. The compound according to claim 1, wherein X and Z areN and Y is C, in free or salt form.
 9. The compound according to claim8, wherein R₆ and R₈ do not exist and R₇ is C₁₋₄alkyl, in free or saltform.
 10. The compound according to claim 8, wherein R₆ and R₈ do notexist and R₇ is methyl, in free or salt form.
 11. The compound accordingto claim 1, wherein: (i) R₁ is —NH(R₂), wherein R₂ is phenyl optionallysubstituted with halo; (ii) At least one of X, Y and Z is N; (iii) R₃,R₄ and R₅ are independently H or C₁₋₄alkyl; or R₃ is H and R₄ and R₅together form a tri-methylene bridge, (iv) R₆, R₇ and R₈ areindependently H or C₁₋₄alkyl; (v) Provided that when X, Y and/or Z areN, then R₆, R₇ and/or R₈, respectively, are not present; and when X, Yand Z are all C, then at least one of R₆, R₇ or R₈ is —S(O)₂—NH₂ orpyrid-2-yl substituted with hydroxy, in free or salt form.
 12. Thecompound according to claim 11, wherein R₃ and R₄ are independentlyC₁₋₄alkyl and R₅ is H, in free or salt form.
 13. The compound accordingto claim 11, wherein R₃ and R₄ are both methyl and R₅ is H, in free orsalt form.
 14. The compound according to claim 11, wherein X and Z areN, Y is C and R₇ is C₁₋₄alkyl, in free or salt form.
 15. The compoundaccording to claim 1 selected from the group consisting of:3-((4-fluorophenyl)amino)-5,7,7-trimethyl-2-((2-methylpyrimidin-5-yl)methyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one;3-((4-fluorophenyl)amino)-5,7,7-trimethyl-2-(pyridin-3-ylmethyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one;3-((4-fluorophenyl)amino)-5,7,7-trimethyl-2-(pyridin-4-ylmethyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one;(6aR,9aS)-2-(4-(6-hydroxypyridin-2-yl)benzyl)-5-methyl-3-(phenylamino)-5,6a,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one;4-(((6aR,9aS)-5-methyl-4-oxo-3-(phenylamino)-4,5,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-2(6aH)-yl)methyl)benzenesulfonamide;3-((4-fluorophenyl)amino)-5,7,7-trimethyl-2-((6-methylpyridin-3-yl)methyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one;4-((3-((4-fluorophenyl)amino)-5,7,7-trimethyl-4-oxo-4,5,7,8-tetrahydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-2-yl)methyl)benzenesulfonamide;3-((4-fluorophenyl)amino)-2-(4-(6-hydroxypyridin-2-yl)benzyl)-5,7,7-trimethyl-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one;2-(4-(6-hydroxypyridin-2-yl)benzyl)-3,5,7,7-tetramethyl-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one;3-ethyl-2-(4-(6-hydroxypyridin-2-yl)benzyl)-5,7,7-trimethyl-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one;in free or salt form.
 16. The compound according to claim 1 which is thecompound3-((4-fluorophenyl)amino)-5,7,7-trimethyl-2-((2-methylpyrimidin-5-yl)methyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one,in free or salt form.
 17. The compound according to claim 1 which is thecompound3-((4-fluorophenyl)amino)-5,7,7-trimethyl-2-(pyridin-3-ylmethyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one,in free or salt form.
 18. The compound according to claim 1 which is thecompound3-((4-fluorophenyl)amino)-5,7,7-trimethyl-2-(pyridin-4-ylmethyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one,in free or salt form.
 19. A combination comprising the compoundaccording to claim 1, in free or pharmaceutically acceptable salt form,and one or more other therapeutic agents useful for the treatment ofcardiovascular disorders, a neutral endopeptidase (NEP or Neprilysin)inhibitor and/or a phosphodiesterase 5 (PDE5) inhibitor), in free orpharmaceutically acceptable salt form.
 20. A pharmaceutical compositioncomprising the compound according to claim 1, in combination orassociation with a pharmaceutically acceptable diluents or carrier. 21.A method for the treatment or prophylaxis of a disease or disorder whichmay be ameliorated by modulating cGMP/PKG-dependent signaling pathwayscomprising administering to a patient in need thereof an effectiveamount of the compound according to claim
 1. 22. The method according toclaim 21, wherein the disease or disorder is a cardiovascular disorder.23. The method according to claim 21 wherein disease or disorder isselected from a group consisting of: hypertrophy, atherosclerosis,myocardial infarction, congestive heart failure, angina, hypertension,essential hypertension, pulmonary hypertension, pulmonary arterialhypertension, secondary pulmonary hypertension, isolated systolichypertension, hypertension associated with diabetes, hypertensionassociated with atherosclerosis, renovascular hypertension.
 24. Themethod according to claim 21, wherein the disease or disorder is cardiachypertrophy.
 25. The method according to claim 1, further comprisingadministering a PDE5 inhibitor, in free or pharmaceutically acceptablesalt form.
 26. The method according to claim 21, wherein the disease ordisorder is a stroke.
 27. The method according to claim 26, wherein thestroke is characterized as a transient ischemic attack.
 28. The methodaccording to claim 21, wherein cardiovascular disease or disorder thatis associated with a muscular dystrophy.
 29. The method according toclaim 28, wherein the disease and disorder is a cardiovasculardysfunction which results from a muscular dystrophy selected from thegroup consisting of: Duchenne muscular dystrophy, Becker musculardystrophy, limb-girdle muscular dystrophy, myotonic dystrophy, andEmery-Dreifuss muscular dystrophy.
 30. The method according to claim 21,wherein the disease or disorder is renal failure, fibrosis, aninflammatory disease or disorder, vascular remodeling and a connectivetissue disease or disorder.
 31. (canceled)
 32. (canceled)